Glyphosate-n-acetyltransferase (glyat) sequences and methods of use

ABSTRACT

Compositions and methods comprising polynucleotides and polypeptides having glyphosate-N-acetyltransferase (GLYAT) activity are provided. In specific embodiments, the sequence has an improved property, such as, but not limited to, an improved specificity for glyphosate when compared to an appropriate control resulting in decreased off target acetylation of, e.g. an amino acid such as aspartate. Further provided are nucleic acid constructs, plants, plant cells, explants, seeds and grain having the GLYAT sequences. Various methods of employing the GLYAT sequences are provided. Such methods include methods for producing a glyphosate tolerant plant, plant cell, explant or seed and methods of controlling weeds in a field containing a crop employing the plants and/or seeds disclosed herein.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/892,663, filed Oct. 18, 2013, which is incorporated by reference inits entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named20141008_BB2152PCT_SeqListing.txt created on Oct. 8, 2014 and having asize of 223 kilobytes and is filed concurrently with the specification.The sequence listing contained in this ASCII formatted document is partof the specification and is herein incorporated by reference in itsentirety.

FIELD

The field relates to the field of molecular biology. More specifically,it pertains to sequences that confer tolerance to glyphosate.

BACKGROUND

In the commercial production of crops, it is desirable to easily andquickly eliminate unwanted plants (i.e., “weeds”) from a field of cropplants. A herbicide treatment could be applied to an entire field thatwould eliminate only the unwanted plants while leaving the crop plantsunharmed. One such treatment system would involve the use of crop plantswhich are tolerant to an herbicide so that when the herbicide wassprayed on a field of herbicide-tolerant crop plants, the crop plantswould continue to thrive while non-herbicide-tolerant weeds were killedor severely damaged.

Crop tolerance to specific herbicides can be conferred by engineeringgenes into crops which encode appropriate herbicide metabolizing enzymesand/or insensitive herbicide targets. In some cases these enzymes, andthe nucleic acids that encode them, originate in a plant. In othercases, they are derived from other organisms, such as microbes. Indeed,transgenic plants have been engineered to express a variety of herbicidetolerance genes from a variety of organisms.

While a number of herbicide-tolerant crop plants are presentlycommercially available, improvements in every aspect of crop production,weed control options, extension of residual weed control, andimprovement in crop yield are continuously in demand. Particularly, dueto local and regional variation in dominant weed species as well aspreferred crop species, a continuing need exists for customized systemsof crop protection and weed management which can be adapted to the needsof a particular region, geography, and/or locality. A continuing needtherefore exists for compositions and methods of crop protection andweed management.

SUMMARY

Compositions and methods comprising polynucleotides and polypeptideshaving glyphosate-N-acetyltransferase (GLYAT) activity are provided. Inspecific embodiments, the sequence has an improved property, such as,but not limited to, an improved specificity for glyphosate when comparedto an appropriate control resulting in decreased non-specificacetylation. Further provided are nucleic acid constructs, plants, plantcells, explants, seeds and grain having the GLYAT sequences.

Various methods of employing the GLYAT sequences are provided. Suchmethods include methods for producing a glyphosate tolerant organisms,plant, plant cell, explant or seed and methods of controlling weeds in afield containing a crop employing the plants and/or seeds.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the N-acetylation of glyphosate catalyzed by aglyphosate-N-acetyltransferase (“GLYAT”).

FIG. 2 shows the structure of glyphosate acetyltransferase. Light coloron ribbon model indicates where substitutions occur in high specificityvariants. Stick model of AcetylCoA and substrate are shown in the activesite cleft. Helices (H) and beta strands (B) mentioned in the text arelabeled.

DETAILED DESCRIPTION

The present disclosure makes reference to the accompanying drawings, inwhich some, but not all embodiments and modifications thereof are shown.Indeed, the embodiments may exist in many different forms and should notbe construed as limited to those set forth herein; rather, theembodiments are provided such that this disclosure will satisfyapplicable legal requirements. Like numbers refer to like elementsthroughout.

Other embodiments and modifications thereof will come to mind to oneskilled in the art to which this disclosure pertains having the benefitof the teachings presented in the foregoing descriptions and theassociated drawings. Therefore, it is to be understood that thedisclosure is not to be limited to the specific embodiments disclosedherein and that modifications and other embodiments are intended to beincluded within the scope of the appended claims. Although specificterms are employed herein, they are used in a generic and descriptivesense only and not for purposes of limitation.

I. Compositions

A. Glyphosate-N-acetyltransferase (GLYAT) Polynucleotides andPolypeptides

As used herein, a “glyphosate-N-acetyltransferase” or “GLYAT”polypeptide or enzyme comprises a polypeptide which hasglyphosate-N-acetyltransferase activity (“GLYAT” activity), i.e., theability to catalyze the acetylation of the secondary amine ofglyphosate, as depicted in FIG. 1. In specific embodiments, apolypeptide having glyphosate-N-acetyltransferase activity can transferthe acetyl group from acetyl CoA to the secondary amine of glyphosate.In addition, one or more GLYAT polypeptides transfer the propionyl groupof propionyl CoA to the secondary amine of glyphosate. One or more GLYATpolypeptides are also capable of catalyzing the acetylation ofglyphosate analogs and/or glyphosate metabolites, e.g.,aminomethylphosphonic acid. In addition, one or more GLYATs are alsoable to transfer the propionyl group of propionyl CoA to glyphosate,indicating that GLYAT is also an acyl transferase. As discussed infurther detail elsewhere herein, the use of fragments and variants ofGLYAT polynucleotides and polypeptides encoded thereby is alsoencompassed by the present disclosure.

Various methods and compositions are provided which employpolynucleotides and polypeptides having GLYAT activity. Such GLYATpolypeptides include those set forth in any one of SEQ ID NO: 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, and/or 172 and biologically active variants andfragments thereof. Further provided are the polynucleotides encodingthese various polypeptides and active variants and fragments thereof.

Further provided are a novel class of GLYAT polypeptides andpolynucleotides encoding the same. Specifically, GLYAT polypeptides, andpolynucleotides encoding the same, are provided which comprise at leastone motif as set forth in SEQ ID NO:174. In further embodiments, theGLYAT polypeptides, and polynucleotides encoding the same, whichcomprise the motif set forth in SEQ ID NO:174, further comprises orencodes a sequence having at least 60%, 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to any GLYAT sequence including any one ofSEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, and/or 172, and/or 173 orany GLYAT sequence found in U.S. Pat. Nos. 7,863,503 and 7,666,643, bothof which are herein incorporated by reference.

Further provided are GLYAT polypeptides, and polynucleotides encodingthe same, wherein the polypeptide has GLYAT activity and, wherein a) theamino acid residue in the encoded polypeptide that corresponds to aminoacid position 3 of SEQ ID NO:2 comprises a serine or a cysteine; b) theamino acid residue in the encoded polypeptide that corresponds to aminoacid position 6 of SEQ ID NO:2 comprises methionine; c) the amino acidresidue in the encoded polypeptide that corresponds to amino acidposition 8 of SEQ ID NO:2 comprises glycine; d) the amino acid residuein the encoded polypeptide that corresponds to amino acid position 23 ofSEQ ID NO:2 comprises glycine, arginine, or lysine; e) the amino acidresidue in the encoded polypeptide that corresponds to amino acidposition 25 of SEQ ID NO:2 comprises serine; f) the amino acid residuein the encoded polypeptide that corresponds to amino acid position 26 ofSEQ ID NO:2 comprises phenylalanine; g) the amino acid residue in theencoded polypeptide that corresponds to amino acid position 27 of SEQ IDNO:2 comprises serine or arginine; h) the amino acid residue in theencoded polypeptide that corresponds to amino acid position 28 of SEQ IDNO:2 comprises arginine or lysine; i) the amino acid residue in theencoded polypeptide that corresponds to amino acid position 29 of SEQ IDNO:2 comprises isoleucine; j) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 31 of SEQ ID NO:2comprises tryptophan; k) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 39 of SEQ ID NO:2comprises cysteine; l) the amino acid residue in the encoded polypeptidethat corresponds to amino acid position 41 of SEQ ID NO:2 comprisesserine; m) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 42 of SEQ ID NO:2 compriseshistidine; n) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 46 of SEQ ID NO:2 comprises cysteine;o) the amino acid residue in the encoded polypeptide that corresponds toamino acid position 48 of SEQ ID NO:2 comprises alanine; p) the aminoacid residue in the encoded polypeptide that corresponds to amino acidposition 58 of SEQ ID NO:2 comprises glycine; q) the amino acid residuein the encoded polypeptide that corresponds to amino acid position 59 ofSEQ ID NO:2 comprises cysteine; r) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 60 of SEQ ID NO:2comprises leucine; s) the amino acid residue in the encoded polypeptidethat corresponds to amino acid position 63 of SEQ ID NO:2 comprisesserine or arginine; t) the amino acid residue in the encoded polypeptidethat corresponds to amino acid position 67 of SEQ ID NO:2 comprisescysteine; u) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 68 of SEQ ID NO:2 comprises valine;v) the amino acid residue in the encoded polypeptide that corresponds toamino acid position 75 of SEQ ID NO:2 comprises alanine; w) the aminoacid residue in the encoded polypeptide that corresponds to amino acidposition 79 of SEQ ID NO:2 comprises glycine; x) the amino acid residuein the encoded polypeptide that corresponds to amino acid position 83 ofSEQ ID NO:2 comprises asparagine; y) the amino acid residue in theencoded polypeptide that corresponds to amino acid position 85 of SEQ IDNO:2 comprises arginine; z) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 87 of SEQ ID NO:2comprises alanine; aa) the amino acid residue in the encoded polypeptidethat corresponds to amino acid position 88 of SEQ ID NO:2 comprisesarginine; bb) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 89 of SEQ ID NO:2 comprises arginineor asparagine; cc) the amino acid residue in the encoded polypeptidethat corresponds to amino acid position 90 of SEQ ID NO:2 comprisesvaline; dd) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 91 of SEQ ID NO:2 comprisesmethionine; ee) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 95 of SEQ ID NO:2 comprisesglutamine; ff) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 96 of SEQ ID NO:2 comprises asparticacid; gg) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 98 of SEQ ID NO:2 comprisesmethionine; hh) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 99 of SEQ ID NO:2 comprises valine,alanine, or lysine; ii) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 100 of SEQ ID NO:2comprises alanine; jj) the amino acid residue in the encoded polypeptidethat corresponds to amino acid position 101 of SEQ ID NO:2 comprisesalanine, cysteine, leucine, or isoleucine; kk) the amino acid residue inthe encoded polypeptide that corresponds to amino acid position 105 ofSEQ ID NO:2 comprises threonine; ll) the amino acid residue in theencoded polypeptide that corresponds to amino acid position 109 of SEQID NO:2 comprises phenylalanine or glutamine; mm) the amino acid residuein the encoded polypeptide that corresponds to amino acid position 112of SEQ ID NO:2 comprises valine, leucine, or methionine; nn) the aminoacid residue in the encoded polypeptide that corresponds to amino acidposition 125 of SEQ ID NO:2 comprises serine; oo) the amino acid residuein the encoded polypeptide that corresponds to amino acid position 128of SEQ ID NO:2 comprises glycine, threonine, alanine, arginine, orcysteine; pp) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 130 of SEQ ID NO:2 comprisestryptophan; qq) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 131 of SEQ ID NO:2 comprisesglutamine or arginine; rr) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 132 of SEQ ID NO:2comprises tyrosine; ss) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 133 of SEQ ID NO:2comprises lysine; tt) the amino acid residue in the encoded polypeptidethat corresponds to amino acid position 137 of SEQ ID NO:2 comprisesglutamic acid, alanine, arginine, or serine; and/or uu) the amino acidresidue in the encoded polypeptide that corresponds to amino acidposition 142 of SEQ ID NO:2 comprises valine or cysteine. In stillfurther embodiments, the GLYAT polypeptide described above furthercomprises at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% toany one of SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, and/or 173.

The GLYAT polypeptides and active variants and fragments thereofdisclosed herein may have improved enzymatic activity when compared topreviously identified GLYAT polypeptides. The GLYAT polypeptidesdisclosed herein can have a lower capacity for acetylation of an aminoacid, such as aspartate, when compared to previously known GLYATenzymes, while still retaining the ability to acetylate glyphosate. Forexample, the GLYAT polypeptides or active variants thereof can have adecrease in k_(cat)/K_(M) for aspartate. By “decrease” is intended anystatistically significant reduction in measured activity when comparedto an appropriate control. In some embodiments, an appropriate controlis a previously known GLYAT sequence, such as that set forth in SEQ IDNO:2. In some embodiments, the decrease in the k_(cat)/K_(M) foraspartate when compared to SEQ ID NO:2 can comprise about a 1, 3, 5, 6,7, 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50 fold or greater reduction in the k_(cat)/K_(M) for aspartate.In still further embodiments, a decrease in the k_(cat)/K_(M) foraspartate can include, for example, a k_(cat)/K_(M) for aspartate ofless than about 5.0, 1.0, 1.0, 0.5, 0.1, 0.05, 0.01 min⁻¹mM⁻¹, or less.

In still further embodiments, the GLYAT polypeptides having a lowercapacity for acetylation of aspartate also have an improved specificityfor acetylating glyphosate. By “improved specificity for glyphosate” isintended the k_(cat)/K_(M) for aspartate divided by the k_(cat)/K_(M)for glyphosate, expressed as a percent, is decreased when compared to anappropriate control. In some embodiments, an appropriate control is apreviously known GLYAT sequence, such as that set forth in SEQ ID NO:2.In some embodiments, the improved specificity for glyphosate whencompared to SEQ ID NO:2 comprises about a 1, 5, 10, 20, 30, 40, 50, 100or more fold reduction in[(k_(cat)/K_(M))_(asp)/(k_(cat)/K_(M))_(gly)]×100. A decrease in the[(k_(cat)/KM)_(asp)/(k_(cat)/K_(M))_(gly)]×100 can include, for example,a value of less than about 0.01%.

While the various GLYAT sequences and active variants and fragmentsthereof disclosed herein can have any one or more of the characteristicsdiscussed above, each of the GLYAT sequences disclosed herein and activevariants or fragments thereof also have “glyphosate-N-acetyltransferaseactivity” or “GLYAT” activity. Enzymatic activity can be characterizedusing the conventional kinetic parameters k_(cat), K_(M), andk_(cat)/K_(M). k_(cat) is the number of moles of substrate transformedper minute per mole of enzyme under optimal conditions, which includessaturating substrate concentration; K_(M) is a measure of the affinityof the GLYAT enzyme for its substrates (e.g., acetyl CoA, propionyl CoAand glyphosate); and k_(cat)/K_(M) is a measure of catalytic efficiencythat takes both substrate affinity and catalytic rate into account.k_(cat)/K_(M) is relevant in the situation where the concentration of asubstrate is at least partially rate-limiting. In general, a GLYAT witha higher k_(cat) or k_(cat)/K_(M) is a more efficient catalyst thananother GLYAT with a lower k_(cat) or k_(cat)/K_(M). A GLYAT with alower K_(M) (for glyphosate) is a more efficient catalyst than anotherGLYAT with a higher K_(M). Thus, to determine whether one GLYAT is moreeffective than another, one can compare kinetic parameters for the twoenzymes. The relevance of k_(cat), k_(cat)/K_(M) and K_(M) will varydepending upon the context in which the GLYAT will be expected tofunction, e.g., the anticipated effective concentration of glyphosaterelative to the K_(M) for glyphosate. GLYAT activity can also becharacterized in terms of any of a number of functional characteristics,including but not limited to stability, susceptibility to inhibition, oractivation by other molecules.

Thus, for example, the GLYAT polypeptide may have a lower K_(M) forglyphosate, for example, less than 1 mM, 0.9 mM, 0.8 mM, 0.7 mM, 0.6 mM,0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM, 0.1 mM, 0.05 mM, or less. The GLYATpolypeptide may have a higher k_(cat) for glyphosate than previouslyidentified enzymes, for example, a k_(cat) of at least 500 min⁻¹, 1000min⁻¹, 1100 min⁻¹, 1200 min⁻¹, 1250 min⁻¹, 1300 min⁻¹, 1400 min⁻¹, 1500min⁻¹, 1600 min⁻¹, 1700 min⁻¹, 1800 min⁻¹, 1900 min⁻¹, or 2000 min⁻¹ orhigher. GLYAT polypeptides as described in this disclosure may have ahigher k_(cat)/K_(M) for glyphosate than previously identified enzymes,for example, a k_(cat)/K_(M) of at least 1000 mM⁻¹min⁻¹, 2000 mM⁻¹min⁻¹,3000 mM⁻¹min⁻¹, 4000 mM⁻¹min⁻¹, 5000 mM⁻¹min⁻¹, 6000 mM⁻¹min⁻¹, 7000mM⁻¹min⁻¹, or 8000 mM⁻¹min⁻¹, or higher. The activity of GLYAT enzymesis affected by, for example, pH and salt concentration; and appropriateassay methods and conditions are known in the art (see, e.g.,WO2005012515). Such improved enzymes may find particular use in methodsof growing a crop in a field where the crop exhibits increased croptolerance against a particular herbicide or combination of herbicidesand/or other agricultural chemicals.

As used herein, an “isolated” or “purified” polynucleotide orpolypeptide, or biologically active portion thereof, is substantially oressentially free from components that normally accompany or interactwith the polynucleotide or polypeptide as found in its naturallyoccurring environment. Thus, an isolated or purified polynucleotide orpolypeptide is substantially free of other cellular material or culturemedium when produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.Optimally, an “isolated” polynucleotide is free of sequences (optimallyprotein encoding sequences) that naturally flank the polynucleotide(i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) inthe genomic DNA of the organism from which the polynucleotide isderived. For purposes of this disclosure, “isolated” or “recombinant”when used to refer to nucleic acid molecules excludes isolatedunmodified chromosomes. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A polypeptide that is substantially free ofcellular material includes preparations of polypeptides having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the polypeptide of the disclosure or a biologically active portionthereof is recombinantly produced, optimally culture medium representsless than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemicalprecursors or non-protein-of-interest chemicals.

As used herein, a “recombinant” polynucleotide comprises a combinationof two or more chemically linked nucleic acid segments which are notfound directly joined in nature. By “directly joined” is intended thetwo nucleic acid segments are immediately adjacent and joined to oneanother by a chemical linkage. In specific embodiments, the recombinantpolynucleotide comprises a polynucleotide of interest or active variantor fragment thereof such that an additional chemically linked nucleicacid segment is located either 5′, 3′ or internal to the polynucleotideof interest. Alternatively, the chemically-linked nucleic acid segmentof the recombinant polynucleotide can be formed by the deletion of asequence. The additional chemically linked nucleic acid segment or thesequence deleted to join the linked nucleic acid segments can be of anylength, including for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 orgreater nucleotides. Various methods for making such recombinantpolynucleotides are disclosed herein, including, for example, bychemical synthesis or by the manipulation of isolated segments ofpolynucleotides by genetic engineering techniques. In specificembodiments, the recombinant polynucleotide can comprise a recombinantDNA sequence or a recombinant RNA sequence.

A “recombinant polynucleotide construct” comprises two or more operablylinked nucleic acid segments which are not found operably linked innature. Non-limiting examples of recombinant polynucleotide constructsinclude a polynucleotide of interest or active variant or fragmentthereof operably linked to heterologous sequences which aid in theexpression, autologous replication, and/or genomic insertion of thesequence of interest. Such heterologous and operably linked sequencesinclude, for example, promoters, termination sequences, enhancers, etc,or any component of an expression cassette; a plasmid, cosmid, virus,autonomously replicating sequence, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA nucleotide sequence;and/or sequences that encode heterologous polypeptides.

A “recombinant polypeptide” comprises a combination of two or morechemically linked amino acid segments which are not found directlyjoined in nature. In specific embodiments, the recombinant polypeptidecomprises an additional chemically linked amino acid segment that islocated either at the N-terminal, C-terminal or internal to therecombinant polypeptide. Alternatively, the chemically-linked amino acidsegment of the recombinant polypeptide can be formed by deletion of atleast one amino acid. The additional chemically linked amino acidsegment or the deleted chemically linked amino acid segment can be ofany length, including for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20or amino acids.

B. Active Fragments and Variants of GLYAT Sequences

Methods and compositions are provided which employ polynucleotides andpolypeptides having glyphosate-N-acetyltransferase activity. Moreover,any given variant or fragment of a GLYAT sequence may further comprisean improved specificity for glyphosate when compared to an appropriatecontrol resulting in decreased non-specific acetylation of, e.g. anamino acid such as aspartate.

i. Polynucleotide and Polypeptide Fragments

Fragments and variants of GLYAT polynucleotides and polypeptides arealso encompassed by the present disclosure. By “fragment” is intended aportion of the polynucleotide or a portion of the amino acid sequenceand hence protein encoded thereby. Fragments of a polynucleotide mayencode protein fragments that retain GLYAT activity, and in specificembodiments, can further comprise an improved specificity for glyphosatewhen compared to an appropriate control resulting in decreasednon-specific acetylation of, e.g. an amino acid such as aspartate.Alternatively, fragments of a polynucleotide that are useful ashybridization probes or PCR primers generally do not encode fragmentproteins retaining biological activity. In specific embodiments, afragment of a recombinant polynucleotide or a recombinant polynucleotideconstruct comprises at least one junction of the two or more chemicallylinked or operably linked nucleic acid segments which are not founddirectly joined in nature. Thus, fragments of a nucleotide sequence mayrange from at least about 20 nucleotides, about 50 nucleotides, about100 nucleotides, and up to the full-length polynucleotide encoding theGLYAT polypeptides. A fragment of a GLYAT polynucleotide that encodes abiologically active portion of a GLYAT protein of the disclosure willencode at least 25, 50, 75, 100, 125, 147 contiguous amino acids, or upto the total number of amino acids present in a full-length GLYATpolypeptide.

Thus, a fragment of a GLYAT polynucleotide may encode a biologicallyactive portion of a GLYAT polypeptide, or it may be a fragment that canbe used as a hybridization probe or PCR primer using methods disclosedbelow. A biologically active portion of a GLYAT polypeptide can beprepared by isolating a portion of one of the GLYAT polynucleotides,expressing the encoded portion of the GLYAT polypeptides (e.g., byrecombinant expression in vitro), and assessing the activity of theGLYAT portion of the GLYAT protein. Polynucleotides that are fragmentsof a GLYAT nucleotide sequence comprise at least 16, 20, 50, 75, 100,150, 200, 250, 300, 350, 400, 450 contiguous nucleotides, or up to thenumber of nucleotides present in a full-length GLYAT polynucleotidedisclosed herein.

Fragments of a polypeptide may encode protein fragments that retainGLYAT activity, and in specific embodiments, can further comprise animproved specificity for glyphosate when compared to an appropriatecontrol resulting in decreased non-specific acetylation of, e.g. anamino acid such as aspartate. A fragment of a GLYAT polypeptidedisclosed herein will encode at least 25, 50, 75, 100, 125, 147contiguous amino acids, or up to the total number of amino acids presentin a full-length GLYAT polypeptide. In specific embodiments, suchpolypeptide fragments are active fragments, and in still otherembodiments, the polypeptide fragment comprises a recombinantpolypeptide fragment. As used herein, a fragment of a recombinantpolypeptide comprises at least one of a combination of two or morechemically linked amino acid segments which are not found directlyjoined in nature.

ii. Polynucleotide and Polypeptide Variants

“Variant” protein is intended to mean a protein derived from the proteinby deletion (i.e., truncation at the 5′ and/or 3′ end) and/or a deletionor addition of one or more amino acids at one or more internal sites inthe native protein and/or substitution of one or more amino acids at oneor more sites in the native protein. Variant proteins encompassed arebiologically active, that is they continue to possess the desiredbiological activity, that is, have GLYAT activity. Moreover, any givenvariant or fragment may further comprise an improved specificity forglyphosate when compared to an appropriate control resulting indecreased non-specific acetylation of, e.g. an amino acid such asaspartate. Such variants may result from, for example, geneticpolymorphism or from human manipulation.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a polynucleotide having a deletion(i.e., truncations) at the 5′ and/or 3′ end and/or a deletion and/oraddition of one or more nucleotides at one or more internal sites withinthe native polynucleotide and/or a substitution of one or morenucleotides at one or more sites in the native polynucleotide. As usedherein, a “native” polynucleotide or polypeptide comprises a naturallyoccurring nucleotide sequence or amino acid sequence, respectively. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the GLYAT polypeptides of the disclosure. Naturallyoccurring variants such as these can be identified with the use ofwell-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant polynucleotides also include synthetically derivedpolynucleotides, such as those generated, for example, by usingsite-directed mutagenesis or gene synthesis but which still encode aGLYAT polypeptide.

Biologically active variants of a GLYAT polypeptide (and thepolynucleotide encoding the same) will have at least about 85%, 90%,91%, 92%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%,98%, 98.5%, 99%, 99.5%, or more sequence identity to the polypeptide ofany one of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, and/or 172 asdetermined by sequence alignment programs and parameters describedelsewhere herein.

In specific embodiments, the biologically active variants of a GLYATpolypeptide (and polynucleotide encoding the same) will have at least94.5% sequence identity to the full length of SEQ ID NO:3; at least96.6% sequence identity to the full length of SEQ ID NO:4; at least93.8% sequence identity to the full length of SEQ ID NO:5; at least92.4% sequence identity to the full length of SEQ ID NO:6; at least91.8% sequence identity to the full length of SEQ ID NO:7; at least93.1% sequence identity to the full length of SEQ ID NO:8; at least93.8% sequence identity to the full length of SEQ ID NO:9; at least93.8% sequence identity to the full length of SEQ ID NO:10; at least93.1% sequence identity to the full length of SEQ ID NO:11; at least92.5% sequence identity to the full length of SEQ ID NO:12; at least93.2% sequence identity to the full length of SEQ ID NO:13; at least93.2% sequence identity to the full length of SEQ ID NO:14; at least95.9% sequence identity to the full length of SEQ ID NO:15; at least92.5% sequence identity to the full length of SEQ ID NO:16; at least90.4% sequence identity to the full length of SEQ ID NO:17; at least93.2% sequence identity to the full length of SEQ ID NO:18; at least93.8% sequence identity to the full length of SEQ ID NO:19; at least92.5% sequence identity to the full length of SEQ ID NO:20; at least95.9% sequence identity to the full length of SEQ ID NO:21; at least91.7% sequence identity to the full length of SEQ ID NO:22; at least92.5% sequence identity to the full length of SEQ ID NO:23; at least90.4% sequence identity to the full length of SEQ ID NO:24; at least89.7% sequence identity to the full length of SEQ ID NO:25; at least92.4% sequence identity to the full length of SEQ ID NO:26; at least91.7% sequence identity to the full length of SEQ ID NO:27; at least87.6% sequence identity to the full length of SEQ ID NO:28; at least88.3% sequence identity to the full length of SEQ ID NO:29; at least 89%sequence identity to the full length of SEQ ID NO:30; at least 87.6%sequence identity to the full length of SEQ ID NO:31; at least 91.7%sequence identity to the full length of SEQ ID NO:32; at least 89.7%sequence identity to the full length of SEQ ID NO:33; at least 89%sequence identity to the full length of SEQ ID NO:34; at least 86.9%sequence identity to the full length of SEQ ID NO:35; at least 89%sequence identity to the full length of SEQ ID NO:36; at least 89.7%sequence identity to the full length of SEQ ID NO:37; at least 87.6%sequence identity to the full length of SEQ ID NO:38; at least 89.7%sequence identity to the full length of SEQ ID NO:39; at least 89.7%sequence identity to the full length of SEQ ID NO:40; at least 89.7%sequence identity to the full length of SEQ ID NO:41; at least 89.7%sequence identity to the full length of SEQ ID NO:42; at least 89.7%sequence identity to the full length of SEQ ID NO:43; at least 88.3%sequence identity to the full length of SEQ ID NO:44; at least 88.3%sequence identity to the full length of SEQ ID NO:45; at least 89.7%sequence identity to the full length of SEQ ID NO:46; at least 89%sequence identity to the full length of SEQ ID NO:47; at least 88.3%sequence identity to the full length of SEQ ID NO:48; at least 88.3%sequence identity to the full length of SEQ ID NO:49; at least 89.7%sequence identity to the full length of SEQ ID NO:50; at least 89.7%sequence identity to the full length of SEQ ID NO:51; at least 88.3%sequence identity to the full length of SEQ ID NO:52; at least 89%sequence identity to the full length of SEQ ID NO:53; at least 87.6%sequence identity to the full length of SEQ ID NO:54; at least 91.7%sequence identity to the full length of SEQ ID NO:55; at least 87.6%sequence identity to the full length of SEQ ID NO:56; at least 91%sequence identity to the full length of SEQ ID NO:57; at least 89%sequence identity to the full length of SEQ ID NO:58; at least 90.3%sequence identity to the full length of SEQ ID NO:59; at least 89%sequence identity to the full length of SEQ ID NO:60; at least 87.6%sequence identity to the full length of SEQ ID NO:61; at least 89.7%sequence identity to the full length of SEQ ID NO:62; at least 89%sequence identity to the full length of SEQ ID NO:63; at least 89%sequence identity to the full length of SEQ ID NO:64; at least 90.3%sequence identity to the full length of SEQ ID NO:65; at least 89.7%sequence identity to the full length of SEQ ID NO:66; at least 89%sequence identity to the full length of SEQ ID NO:67; at least 88.3%sequence identity to the full length of SEQ ID NO:68; at least 90.3%sequence identity to the full length of SEQ ID NO:69; at least 89%sequence identity to the full length of SEQ ID NO:70; at least 89%sequence identity to the full length of SEQ ID NO:71; at least 88.3%sequence identity to the full length of SEQ ID NO:72; at least 89%sequence identity to the full length of SEQ ID NO:73; at least 89.7%sequence identity to the full length of SEQ ID NO:74; at least 88.3%sequence identity to the full length of SEQ ID NO:75; at least 88.3%sequence identity to the full length of SEQ ID NO:76; at least 89%sequence identity to the full length of SEQ ID NO:77; at least 91%sequence identity to the full length of SEQ ID NO:78; at least 90.3%sequence identity to the full length of SEQ ID NO:79; at least 90.3%sequence identity to the full length of SEQ ID NO:80; at least 89%sequence identity to the full length of SEQ ID NO:81; at least 89%sequence identity to the full length of SEQ ID NO:82; at least 89.7%sequence identity to the full length of SEQ ID NO:83; at least 89.7%sequence identity to the full length of SEQ ID NO:84; at least 89%sequence identity to the full length of SEQ ID NO:85; at least 89.7%sequence identity to the full length of SEQ ID NO:86; at least 88.3%sequence identity to the full length of SEQ ID NO:87; at least 90.3%sequence identity to the full length of SEQ ID NO:88; at least 90.3%sequence identity to the full length of SEQ ID NO:89; at least 89%sequence identity to the full length of SEQ ID NO:90; at least 87.6%sequence identity to the full length of SEQ ID NO:91; at least 89%sequence identity to the full length of SEQ ID NO:92; at least 89.7%sequence identity to the full length of SEQ ID NO:93; at least 90.3%sequence identity to the full length of SEQ ID NO:94; at least 89.7%sequence identity to the full length of SEQ ID NO:95; at least 89%sequence identity to the full length of SEQ ID NO:96; at least 89%sequence identity to the full length of SEQ ID NO:97; at least 89%sequence identity to the full length of SEQ ID NO:98; at least 91%sequence identity to the full length of SEQ ID NO:99; at least 88.3%sequence identity to the full length of SEQ ID NO:100; at least 89%sequence identity to the full length of SEQ ID NO:101; at least 91.7%sequence identity to the full length of SEQ ID NO: 102; at least 91.7%sequence identity to the full length of SEQ ID NO: 103; at least 93%sequence identity to the full length of SEQ ID NO: 104; at least 91.7%sequence identity to the full length of SEQ ID NO: 105; at least 91.7%sequence identity to the full length of SEQ ID NO: 106; at least 91.7%sequence identity to the full length of SEQ ID NO:107; at least 91.7%sequence identity to the full length of SEQ ID NO: 108; at least 91.7%sequence identity to the full length of SEQ ID NO:109; at least 91.7%sequence identity to the full length of SEQ ID NO:110; at least 91.7%sequence identity to the full length of SEQ ID NO:111; at least 91.7%sequence identity to the full length of SEQ ID NO:112; at least 91.7%sequence identity to the full length of SEQ ID NO:113; at least 91.7%sequence identity to the full length of SEQ ID NO:114; at least 91.7%sequence identity to the full length of SEQ ID NO:115; at least 92.4%sequence identity to the full length of SEQ ID NO:116; at least 92.4%sequence identity to the full length of SEQ ID NO:117; at least 93.1%sequence identity to the full length of SEQ ID NO:118; at least 91.7%sequence identity to the full length of SEQ ID NO:119; at least 91.7%sequence identity to the full length of SEQ ID NO:120; at least 91.7%sequence identity to the full length of SEQ ID NO:121; at least 91.7%sequence identity to the full length of SEQ ID NO:122; at least 91.7%sequence identity to the full length of SEQ ID NO:123; at least 91.7%sequence identity to the full length of SEQ ID NO:124; at least 91.7%sequence identity to the full length of SEQ ID NO:125; at least 91.7%sequence identity to the full length of SEQ ID NO:126; at least 91.7%sequence identity to the full length of SEQ ID NO:127; at least 91.7%sequence identity to the full length of SEQ ID NO:128; at least 92.4%sequence identity to the full length of SEQ ID NO:129; at least 91.7%sequence identity to the full length of SEQ ID NO:130; at least 91.7%sequence identity to the full length of SEQ ID NO:131; at least 91.7%sequence identity to the full length of SEQ ID NO: 132; at least 91.7%sequence identity to the full length of SEQ ID NO:133; at least 91.7%sequence identity to the full length of SEQ ID NO: 134; at least 91.7%sequence identity to the full length of SEQ ID NO:135; at least 91.7%sequence identity to the full length of SEQ ID NO: 136; at least 91.7%sequence identity to the full length of SEQ ID NO: 137; at least 91.7%sequence identity to the full length of SEQ ID NO: 138; at least 92.4%sequence identity to the full length of SEQ ID NO:139; at least 91.7%sequence identity to the full length of SEQ ID NO:140; at least 91.7%sequence identity to the full length of SEQ ID NO:141; at least 91.7%sequence identity to the full length of SEQ ID NO:142; at least 91.7%sequence identity to the full length of SEQ ID NO:143; at least 91.7%sequence identity to the full length of SEQ ID NO:144; at least 91.7%sequence identity to the full length of SEQ ID NO:145; at least 91.7%sequence identity to the full length of SEQ ID NO:146; at least 91.7%sequence identity to the full length of SEQ ID NO:147; at least 91.7%sequence identity to the full length of SEQ ID NO:148; at least 91.7%sequence identity to the full length of SEQ ID NO:149; at least 92.4%sequence identity to the full length of SEQ ID NO: 150; at least 91.7%sequence identity to the full length of SEQ ID NO:151; at least 91.7%sequence identity to the full length of SEQ ID NO:152; at least 91.7%sequence identity to the full length of SEQ ID NO: 153; at least 91.7%sequence identity to the full length of SEQ ID NO:154; at least 91.7%sequence identity to the full length of SEQ ID NO:155; at least 91%sequence identity to the full length of SEQ ID NO:156; at least 91.7%sequence identity to the full length of SEQ ID NO: 157; at least 91.7%sequence identity to the full length of SEQ ID NO: 158; at least 91.7%sequence identity to the full length of SEQ ID NO: 159; at least 91.7%sequence identity to the full length of SEQ ID NO:160; at least 91.7%sequence identity to the full length of SEQ ID NO:161; at least 91.7%sequence identity to the full length of SEQ ID NO:162; at least 92.4%sequence identity to the full length of SEQ ID NO:163; at least 92.4%sequence identity to the full length of SEQ ID NO:164; at least 91.7%sequence identity to the full length of SEQ ID NO:165; at least 91.7%sequence identity to the full length of SEQ ID NO:166; at least 91.7%sequence identity to the full length of SEQ ID NO:167; at least 91.7%sequence identity to the full length of SEQ ID NO:168; at least 91.7%sequence identity to the full length of SEQ ID NO:169; at least 91.7%sequence identity to the full length of SEQ ID NO:170; at least 91.7%sequence identity to the full length of SEQ ID NO:171; and/or at least91.7% sequence identity to the full length of SEQ ID NO:172; whereinpercent identity is determined using the BLAST alignment used theBLOSUM62 substitution matrix, a gap existence penalty of 11, and a gapextension penalty of 1.

In other embodiments, variants of a GLYAT polypeptide (and thepolynucleotides encoding the same) will have a similarity score of atleast 650, 660, 670, 680, 690, 700, 710, 720, 725, 730, 731, 735, 740,745, 750, 755, 760, 761, 762, 765, 770, 775, 780, 785, 790, 795, 800,805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870,875, 880, 885, 890, 895, 900, 920, 950, 975, 1000, or greater asdetermined by parameters described elsewhere herein to a polynucleotideencoding any one of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, and/or 172.In specific embodiments, the biologically active GLYAT variant (andpolynucleotide encoding the same) comprises a polypeptide havingglyphosate-N-acetyltransferase (GLYAT) activity and further comprisingan amino acid sequence having: a similarity score of at least 760 withSEQ ID NO:3; a similarity score of at least 762 with SEQ ID NO:4; asimilarity score of at least 744 with SEQ ID NO:5; a similarity score ofat least 738 with SEQ ID NO:6; a similarity score of at least 728 withSEQ ID NO:7; a similarity score of at least 746 with SEQ ID NO:8; asimilarity score of at least 747 with SEQ ID NO:9; a similarity score ofat least 740 with SEQ ID NO:10; a similarity score of at least 747 withSEQ ID NO:11; a similarity score of at least 746 with SEQ ID NO:12; asimilarity score of at least 746 with SEQ ID NO:13; a similarity scoreof at least 740 with SEQ ID NO:14; a similarity score of at least 756with SEQ ID NO:15; a similarity score of at least 736 with SEQ ID NO:16;a similarity score of at least 721 with SEQ ID NO:17; a similarity scoreof at least 744 with SEQ ID NO:18; a similarity score of at least 744with SEQ ID NO:19; a similarity score of at least 737 with SEQ ID NO:20;a similarity score of at least 753 with SEQ ID NO:21; a similarity scoreof at least 727 with SEQ ID NO:22; a similarity score of at least 742with SEQ ID NO:23; a similarity score of at least 718 with SEQ ID NO:24;a similarity score of at least 703 with SEQ ID NO:25; a similarity scoreof at least 738 with SEQ ID NO:26; a similarity score of at least 731with SEQ ID NO:27; a similarity score of at least 708 with SEQ ID NO:28;a similarity score of at least 707 with SEQ ID NO:29; a similarity scoreof at least 707 with SEQ ID NO:30; a similarity score of at least 700with SEQ ID NO:31; a similarity score of at least 726 with SEQ ID NO:32;a similarity score of at least 714 with SEQ ID NO:33; a similarity scoreof at least 703 with SEQ ID NO:34; a similarity score of at least 694with SEQ ID NO:35; a similarity score of at least 720 with SEQ ID NO:36;a similarity score of at least 721 with SEQ ID NO:37; a similarity scoreof at least 694 with SEQ ID NO:38; a similarity score of at least 714with SEQ ID NO:39; a similarity score of at least 722 with SEQ ID NO:40;a similarity score of at least 718 with SEQ ID NO:41; a similarity scoreof at least 721 with SEQ ID NO:42; a similarity score of at least 717with SEQ ID NO:43; a similarity score of at least 707 with SEQ ID NO:44;a similarity score of at least 709 with SEQ ID NO:45; a similarity scoreof at least 717 with SEQ ID NO:46; a similarity score of at least 714with SEQ ID NO:47; a similarity score of at least 704 with SEQ ID NO:48;a similarity score of at least 709 with SEQ ID NO:49; a similarity scoreof at least 717 with SEQ ID NO:50; a similarity score of at least 719with SEQ ID NO:51; a similarity score of at least 707 with SEQ ID NO:52;a similarity score of at least 712 with SEQ ID NO:53; a similarity scoreof at least 704 with SEQ ID NO:54; a similarity score of at least 733with SEQ ID NO:55; a similarity score of at least 694 with SEQ ID NO:56;a similarity score of at least 722 with SEQ ID NO:57; a similarity scoreof at least 707 with SEQ ID NO:58; a similarity score of at least 718with SEQ ID NO:59; a similarity score of at least 708 with SEQ ID NO:60;a similarity score of at least 700 with SEQ ID NO:61; a similarity scoreof at least 714 with SEQ ID NO:62; a similarity score of at least 708with SEQ ID NO:63; a similarity score of at least 714 with SEQ ID NO:64;a similarity score of at least 724 with SEQ ID NO:65; a similarity scoreof at least 713 with SEQ ID NO:66; a similarity score of at least 710with SEQ ID NO:67; a similarity score of at least 710 with SEQ ID NO:68;a similarity score of at least 720 with SEQ ID NO:69; a similarity scoreof at least 709 with SEQ ID NO:70; a similarity score of at least 714with SEQ ID NO:71; a similarity score of at least 703 with SEQ ID NO:72;a similarity score of at least 709 with SEQ ID NO:73; a similarity scoreof at least 720 with SEQ ID NO:74; a similarity score of at least 708with SEQ ID NO:75; a similarity score of at least 700 with SEQ ID NO:76;a similarity score of at least 715 with SEQ ID NO:77; a similarity scoreof at least 722 with SEQ ID NO:78; a similarity score of at least 724with SEQ ID NO:79; a similarity score of at least 723 with SEQ ID NO:80;a similarity score of at least 709 with SEQ ID NO:81; a similarity scoreof at least 709 with SEQ ID NO:82; a similarity score of at least 716with SEQ ID NO:83; a similarity score of at least 721 with SEQ ID NO:84;a similarity score of at least 711 with SEQ ID NO:85; a similarity scoreof at least 717 with SEQ ID NO:86; a similarity score of at least 705with SEQ ID NO:87; a similarity score of at least 720 with SEQ ID NO:88;a similarity score of at least 728 with SEQ ID NO:89; a similarity scoreof at least 717 with SEQ ID NO:90; a similarity score of at least 703with SEQ ID NO:91; a similarity score of at least 710 with SEQ ID NO:92;a similarity score of at least 716 with SEQ ID NO:93; a similarity scoreof at least 723 with SEQ ID NO:94; a similarity score of at least 715with SEQ ID NO:95; a similarity score of at least 716 with SEQ ID NO:96;a similarity score of at least 709 with SEQ ID NO:97; a similarity scoreof at least 718 with SEQ ID NO:98; a similarity score of at least 726with SEQ ID NO:99; a similarity score of at least 705 with SEQ IDNO:100; a similarity score of at least 709 with SEQ ID NO:101; asimilarity score of at least 736 with SEQ ID NO:102; a similarity scoreof at least 734 with SEQ ID NO:103; a similarity score of at least 729with SEQ ID NO:104; a similarity score of at least 736 with SEQ IDNO:105; a similarity score of at least 730 with SEQ ID NO:106; asimilarity score of at least 730 with SEQ ID NO:107; a similarity scoreof at least 733 with SEQ ID NO:108; a similarity score of at least 732with SEQ ID NO:109; a similarity score of at least 730 with SEQ IDNO:110; a similarity score of at least 732 with SEQ ID NO:111; asimilarity score of at least 731 with SEQ ID NO:112; a similarity scoreof at least 733 with SEQ ID NO:113; a similarity score of at least 734with SEQ ID NO:114; a similarity score of at least 733 with SEQ IDNO:115; a similarity score of at least 731 with SEQ ID NO:116; asimilarity score of at least 733 with SEQ ID NO:117; a similarity scoreof at least 732 with SEQ ID NO:118; a similarity score of at least 735with SEQ ID NO:119; a similarity score of at least 732 with SEQ IDNO:120; a similarity score of at least 732 with SEQ ID NO:121; asimilarity score of at least 731 with SEQ ID NO:122; a similarity scoreof at least 729 with SEQ ID NO:123; a similarity score of at least 732with SEQ ID NO:124; a similarity score of at least 730 with SEQ IDNO:125; a similarity score of at least 732 with SEQ ID NO:126; asimilarity score of at least 734 with SEQ ID NO:127; a similarity scoreof at least 733 with SEQ ID NO:128; a similarity score of at least 732with SEQ ID NO:129; a similarity score of at least 732 with SEQ IDNO:130; a similarity score of at least 735 with SEQ ID NO:131; asimilarity score of at least 734 with SEQ ID NO:132; a similarity scoreof at least 732 with SEQ ID NO:133; a similarity score of at least 735with SEQ ID NO:134; a similarity score of at least 736 with SEQ IDNO:135; a similarity score of at least 732 with SEQ ID NO:136; asimilarity score of at least 733 with SEQ ID NO:137; a similarity scoreof at least 736 with SEQ ID NO:138; a similarity score of at least 736with SEQ ID NO:139; a similarity score of at least 736 with SEQ IDNO:140; a similarity score of at least 736 with SEQ ID NO:141; asimilarity score of at least 731 with SEQ ID NO:142; a similarity scoreof at least 735 with SEQ ID NO:143; a similarity score of at least 733with SEQ ID NO:144; a similarity score of at least 734 with SEQ IDNO:145; a similarity score of at least 733 with SEQ ID NO:146; asimilarity score of at least 730 with SEQ ID NO:147; a similarity scoreof at least 728 with SEQ ID NO:148; a similarity score of at least 734with SEQ ID NO:149; a similarity score of at least 735 with SEQ IDNO:150; a similarity score of at least 733 with SEQ ID NO:151; asimilarity score of at least 731 with SEQ ID NO:152; a similarity scoreof at least 734 with SEQ ID NO:153; a similarity score of at least 732with SEQ ID NO:154; a similarity score of at least 734 with SEQ IDNO:155; a similarity score of at least 726 with SEQ ID NO:156; asimilarity score of at least 733 with SEQ ID NO:157; a similarity scoreof at least 730 with SEQ ID NO:158; a similarity score of at least 733with SEQ ID NO:159; a similarity score of at least 733 with SEQ IDNO:160; a similarity score of at least 728 with SEQ ID NO:161; asimilarity score of at least 733 with SEQ ID NO:162; a similarity scoreof at least 735 with SEQ ID NO:163; a similarity score of at least 733with SEQ ID NO:164; a similarity score of at least 734 with SEQ IDNO:165; a similarity score of at least 731 with SEQ ID NO:166; asimilarity score of at least 732 with SEQ ID NO:167; a similarity scoreof at least 732 with SEQ ID NO:168; a similarity score of at least 729with SEQ ID NO:169; a similarity score of at least 736 with SEQ IDNO:170; a similarity score of at least 732 with SEQ ID NO:171; and/or asimilarity score of at least 729 with SEQ ID NO:172; wherein saidsimilarity score is generated using the BLAST alignment program, withthe BLOSUM62 substitution matrix, a gap existence penalty of 11, and agap extension penalty of 1.

In still further embodiments, a biologically active variant of a GLYATprotein may differ from that protein by 75, 70, 65, 60, 55, 50, 45, 40,35, 30, 25, 20, 19, 18, 17, 16 amino acid residues, as few as 1-15 aminoacid residues, as few as 1-10, such as 6-10, as few as 10, 9, 8, 7, 6,5, as few as 4, 3, 2, or even 1 amino acid residue.

The GLYAT polypeptide and the active variants and fragments thereof maybe altered in various ways including amino acid substitutions,deletions, truncations, and insertions. Methods for such manipulationsare generally known in the art. For example, amino acid sequencevariants and fragments of the GLYAT proteins can be prepared bymutations in the DNA. Methods for mutagenesis and polynucleotidealterations are well known in the art. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be optimal.

The mutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and optimally will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

In general, methods to modify or alter the host genomic DNA areavailable. For example, a pre-existing GLYAT sequence in a host plantcan be modified or altered in a site-specific fashion using one or moresite-specific engineering system. This includes altering the host DNAsequence or a pre-existing transgenic sequence including regulatoryelements, coding and non-coding sequences. These methods are also usefulin targeting nucleic acids to pre-engineered target recognitionsequences in the genome. As an example, the genetically modified cell orplant described herein, is generated using “custom” or engineeredendonucleases such as meganucleases produced to modify plant genomes(see e.g., WO 2009/114321; Gao et al. (2010) Plant Journal 1:176-187).Another site-directed engineering is through the use of zinc fingerdomain recognition coupled with the restriction properties ofrestriction enzyme. See e.g., Urnov, et al., (2010) Nat Rev Genet.11(9):636-46; Shukla, et al., (2009) Nature 459 (7245):437-41. Atranscription activator-like (TAL) effector-DNA modifying enzyme (TALEor TALEN) is also used to engineer changes in plant genome. See e.g.,US20110145940, Cermak et al., (2011) Nucleic Acids Res. 39(12) and Bochet al., (2009), Science 326(5959): 1509-12. Site-specific modificationof plant genomes can also be performed using the bacterial type IICRISPR (clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated) system. See e.g., Belhaj et al., (2013), PlantMethods 9: 39; The CRISPR/Cas system allows targeted cleavage of genomicDNA guided by a customizable small noncoding RNA. Based on thedisclosure of the FC coding sequences, polypeptide sequences of theorthologs/homologs and the genomic DNA sequences, site-directedmutagenesis can be readily performed to generate plants expressing ahigher level of the endogenous FC polypeptide or an ortholog thereof.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different GLYAT codingsequences can be manipulated to create a new GLYAT possessing thedesired properties. In this manner, libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. For example, using this approach, sequence motifs encoding adomain of interest may be shuffled between the GLYAT sequences disclosedherein and other known GLYAT genes to obtain a new gene coding for aprotein with an improved property of interest, such as an improvement inGLYAT activity and/or an improved specificity for glyphosate whencompared to an appropriate control resulting in decreased non-specificacetylation of, e.g. an amino acid such as aspartate.

D. Sequence Comparisons

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: “referencesequence”, “comparison window”, “sequence identity”, and, “percentsequence identity.”

As used herein, “reference sequence” is a predetermined sequence used asa basis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or gene sequenceor protein sequence.

As used herein, “comparison window” makes reference to a contiguous andspecified segment of a polypeptide sequence, wherein the polypeptidesequence in the comparison window may comprise additions or deletions(i.e., gaps) compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two polypeptides.Generally, the comparison window is at least 5, 10, 15, or 20 contiguousamino acids in length, or it can be 30, 40, 50, 100, or longer. Those ofskill in the art understand that to avoid a high similarity to areference sequence due to inclusion of gaps in the polypeptide sequencea gap penalty is typically introduced and is subtracted from the numberof matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of thedisclosure. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the disclosure. BLASTP proteinsearches can be performed using default parameters. See,blast.ncbi.nlm.nih.gov/Blast.cgi.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. See Altschul et al. (1997) supra. When utilizingBLAST, Gapped BLAST, or PSI-BLAST, the default parameters of therespective programs (e.g., BLASTN for nucleotide sequences, BLASTP forproteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also beperformed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity). When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percent sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percent sequence identity” means the value determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison, andmultiplying the result by 100 to yield the percent sequence identity.

Two sequences are “optimally aligned” when they are aligned forsimilarity scoring using a defined amino acid substitution matrix (e.g.,BLOSUM62), gap existence penalty and gap extension penalty so as toarrive at the highest score possible for that pair of sequences. Aminoacids substitution matrices and their use in quantifying the similaritybetween two sequences are well-known in the art and described, e.g., inDayhoff et al. (1978) “A model of evolutionary change in proteins.” In“Atlas of Protein Sequence and Structure,” Vol. 5, Suppl. 3 (ed. M. O.Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D.C. andHenikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919. TheBLOSUM62 matrix is often used as a default scoring substitution matrixin sequence alignment protocols such as Gapped BLAST 2.0. The gapexistence penalty is imposed for the introduction of a single amino acidgap in one of the aligned sequences, and the gap extension penalty isimposed for each additional empty amino acid position inserted into analready opened gap. The gap existence penalty is imposed for theintroduction of a single amino acid gap in one of the aligned sequences,and the gap extension penalty is imposed for each additional empty aminoacid position inserted into an already opened gap. The alignment isdefined by the amino acids positions of each sequence at which thealignment begins and ends, and optionally by the insertion of a gap ormultiple gaps in one or both sequences, so as to arrive at the highestpossible score. While optimal alignment and scoring can be accomplishedmanually, the process is facilitated by the use of acomputer-implemented alignment algorithm, e.g., gapped BLAST 2.0,described in Altschul et al, (1997) Nucleic Acids Res. 25:3389-3402, andmade available to the public at the National Center for BiotechnologyInformation Website (http://www.ncbi.nlm.nih.gov). Optimal alignments,including multiple alignments, can be prepared using, e.g., PSI-BLAST,available through http://www.ncbi.nlm.nih.gov and described by Altschulet al, (1997) Nucleic Acids Res. 25:3389-3402.

As used herein, similarity score and bit score is determined employingthe BLAST alignment used the BLOSUM62 substitution matrix, a gapexistence penalty of 11, and a gap extension penalty of 1. For the samepair of sequences, if there is a numerical difference between the scoresobtained when using one or the other sequence as query sequences, agreater value of similarity score is selected.

E. Plants and Other Host Cells of Interest

Further provided are engineered host cells that are transduced(transformed or transfected) with one or more GLYAT sequences or activevariants or fragments thereof. The GLYAT polypeptides or variants andfragments thereof can be expressed in any organism, including innon-animal cells such as plants, yeast, fungi, bacteria and the like.Details regarding non-animal cell culture can be found in Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin, Heidelberg, New York); and Atlas and Parks(eds.) The Handbook of Microbiological Media (1993) CRC Press, BocaRaton, Fla.

Plants, plant cells, plant parts and seeds, and grain having the GLYATsequences disclosed herein are also provided. In specific embodiments,the plants and/or plant parts have stably incorporated at least oneheterologous GLYAT polypeptide disclosed herein or an active variant orfragment thereof. Thus, plants, plant cells, plant parts and seeds areprovided which comprise at least one heterologous GLYAT sequences of anyone or more of SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, or abiologically active fragment and/or variant thereof.

In other embodiments, plants, plant cells, plant parts and seeds areprovided which comprise at least one heterologous GLYAT polypeptides (orpolynucleotides encoding the same) which comprise at least one motif asset forth in SEQ ID NO:174. In further embodiments, the plants, plantcells, plant parts and seeds comprise a nucleotide sequence whichencodes the motif set forth in SEQ ID NO:174 and further encodes asequence having at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to any GLYAT sequence including any one of SEQ IDNO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, and/or 173 or any GLYATsequence found in U.S. Pat. Nos. 7,863,503 and 7,666,643, both of whichare herein incorporated by reference.

Further provided are plants, plant cells, plant parts and seedscomprising at least one heterologous GLYAT polypeptide (orpolynucleotide encoding the same), wherein the polypeptide has GLYATactivity and, wherein a) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 3 of SEQ ID NO:2comprises a serine or a cysteine; b) the amino acid residue in theencoded polypeptide that corresponds to amino acid position 6 of SEQ IDNO:2 comprises methionine; c) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 8 of SEQ ID NO:2comprises glycine; d) the amino acid residue in the encoded polypeptidethat corresponds to amino acid position 23 of SEQ ID NO:2 comprisesglycine, arginine, or lysine; e) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 25 of SEQ ID NO:2comprises serine; f) the amino acid residue in the encoded polypeptidethat corresponds to amino acid position 26 of SEQ ID NO:2 comprisesphenylalanine; g) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 27 of SEQ ID NO:2 comprises serine orarginine; h) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 28 of SEQ ID NO:2 comprises arginineor lysine; i) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 29 of SEQ ID NO:2 comprisesisoleucine; j) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 31 of SEQ ID NO:2 comprisestryptophan; k) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 39 of SEQ ID NO:2 comprises cysteine;l) the amino acid residue in the encoded polypeptide that corresponds toamino acid position 41 of SEQ ID NO:2 comprises serine; m) the aminoacid residue in the encoded polypeptide that corresponds to amino acidposition 42 of SEQ ID NO:2 comprises histidine; n) the amino acidresidue in the encoded polypeptide that corresponds to amino acidposition 46 of SEQ ID NO:2 comprises cysteine; o) the amino acid residuein the encoded polypeptide that corresponds to amino acid position 48 ofSEQ ID NO:2 comprises alanine; p) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 58 of SEQ ID NO:2comprises glycine; q) the amino acid residue in the encoded polypeptidethat corresponds to amino acid position 59 of SEQ ID NO:2 comprisescysteine; r) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 60 of SEQ ID NO:2 comprises leucine;s) the amino acid residue in the encoded polypeptide that corresponds toamino acid position 63 of SEQ ID NO:2 comprises serine or arginine; t)the amino acid residue in the encoded polypeptide that corresponds toamino acid position 67 of SEQ ID NO:2 comprises cysteine; u) the aminoacid residue in the encoded polypeptide that corresponds to amino acidposition 68 of SEQ ID NO:2 comprises valine; v) the amino acid residuein the encoded polypeptide that corresponds to amino acid position 75 ofSEQ ID NO:2 comprises alanine; w) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 79 of SEQ ID NO:2comprises glycine; x) the amino acid residue in the encoded polypeptidethat corresponds to amino acid position 83 of SEQ ID NO:2 comprisesasparagine; y) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 85 of SEQ ID NO:2 comprises arginine;z) the amino acid residue in the encoded polypeptide that corresponds toamino acid position 87 of SEQ ID NO:2 comprises alanine; aa) the aminoacid residue in the encoded polypeptide that corresponds to amino acidposition 88 of SEQ ID NO:2 comprises arginine; bb) the amino acidresidue in the encoded polypeptide that corresponds to amino acidposition 89 of SEQ ID NO:2 comprises arginine or asparagine; cc) theamino acid residue in the encoded polypeptide that corresponds to aminoacid position 90 of SEQ ID NO:2 comprises valine; dd) the amino acidresidue in the encoded polypeptide that corresponds to amino acidposition 91 of SEQ ID NO:2 comprises methionine; ee) the amino acidresidue in the encoded polypeptide that corresponds to amino acidposition 95 of SEQ ID NO:2 comprises glutamine; ff) the amino acidresidue in the encoded polypeptide that corresponds to amino acidposition 96 of SEQ ID NO:2 comprises aspartic acid; gg) the amino acidresidue in the encoded polypeptide that corresponds to amino acidposition 98 of SEQ ID NO:2 comprises methionine; hh) the amino acidresidue in the encoded polypeptide that corresponds to amino acidposition 99 of SEQ ID NO:2 comprises valine, alanine, or lysine; ii) theamino acid residue in the encoded polypeptide that corresponds to aminoacid position 100 of SEQ ID NO:2 comprises alanine; jj) the amino acidresidue in the encoded polypeptide that corresponds to amino acidposition 101 of SEQ ID NO:2 comprises alanine, cysteine, leucine, orisoleucine; kk) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 105 of SEQ ID NO:2 comprisesthreonine; ll) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 109 of SEQ ID NO:2 comprisesphenylalanine or glutamine; mm) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 112 of SEQ ID NO:2comprises valine, leucine, or methionine; nn) the amino acid residue inthe encoded polypeptide that corresponds to amino acid position 125 ofSEQ ID NO:2 comprises serine; oo) the amino acid residue in the encodedpolypeptide that corresponds to amino acid position 128 of SEQ ID NO:2comprises glycine, threonine, alanine, arginine, or cysteine; pp) theamino acid residue in the encoded polypeptide that corresponds to aminoacid position 130 of SEQ ID NO:2 comprises tryptophan; qq) the aminoacid residue in the encoded polypeptide that corresponds to amino acidposition 131 of SEQ ID NO:2 comprises glutamine or arginine; rr) theamino acid residue in the encoded polypeptide that corresponds to aminoacid position 132 of SEQ ID NO:2 comprises tyrosine; ss) the amino acidresidue in the encoded polypeptide that corresponds to amino acidposition 133 of SEQ ID NO:2 comprises lysine; tt) the amino acid residuein the encoded polypeptide that corresponds to amino acid position 137of SEQ ID NO:2 comprises glutamic acid, alanine, arginine, or serine;and/or uu) the amino acid residue in the encoded polypeptide thatcorresponds to amino acid position 142 of SEQ ID NO:2 comprises valineor cysteine. In still further embodiments, the GLYAT polypeptidedescribed above further comprises at least 60%, 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% to any one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, and/or 173.

In specific embodiments, the GLYAT sequence or active variants andfragments thereof have GLYAT activity. In further embodiments, thesequence has GLYAT activity and has an improved specificity forglyphosate when compared to an appropriate control resulting indecreased non-specific acetylation of, e.g. an amino acid such asaspartate.

In addition, the plants or organism of interest can comprise multipleGLYAT polynucleotides (i.e., at least 1, 2, 3, 4, 5, 6 or more). It isrecognized that if multiple GLYAT polynucleotides are employed, theGLYAT polynucleotides may encode GLYAT polypeptides having (1) differentkinetic parameters, i.e., a GLYAT variant having a lower K_(M) can becombined with one having a higher k_(cat); (2) different specificity forglyphosate when compared to an appropriate control; and/or (3) differentcapacity for acetylation of an amino acid, such as aspartate, whencompared to an appropriate control.

In specific embodiments, the heterologous polynucleotide in the plant orplant part is operably linked to a constitutive, tissue-preferred, orother promoter for expression in plants.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the disclosure,provided that these parts comprise the introduced polynucleotides.

The GLYAT sequences and active variants and fragments thereof disclosedherein may be used for transformation of any plant species, including,but not limited to, monocots and dicots. Examples of plant species ofinterest include, but are not limited to, corn (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, conifers, turf grasses (including cool seasonalgrasses and warm seasonal grasses).

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing that which is disclosedinclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis), and Poplar and Eucalyptus. In specificembodiments, plants of the present disclosure are crop plants (forexample, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower,peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments,corn and soybean plants are optimal, and in yet other embodiments cornplants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

A “subject plant or plant cell” is one in which genetic alteration, suchas transformation, has been affected as to a gene of interest, or is aplant or plant cell which is descended from a plant or cell so alteredand which comprises the alteration. A “control” or “control plant” or“control plant cell” provides a reference point for measuring changes inphenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or cell, i.e., of the same genotype as the starting material forthe genetic alteration which resulted in the subject plant or cell; (b)a plant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e. with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest; or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

In specific embodiments, the glyphosate tolerant plants express a GLYATpolypeptide, i.e., a polypeptide having glyphosate-N-acetyltransferaseactivity wherein the acetyl group from acetyl CoA is transferred to thesecondary amine of glyphosate. Thus, plants of the disclosure that havebeen treated with glyphosate can contain the metaboliteN-acetylglyphosate (“NAG”). Methods to detect glyphosate and NAG aredescribed for example in U.S. Pat. No. 8,003,398, which is herebyincorporated by reference.

Additional host cells of interest can be a eukaryotic cell, an animalcell, a protoplast, a tissue culture cell, prokaryotic cell, a bacterialcell, such as E. coli, B. subtilis, Streptomyces, Salmonellatyphimurium, a gram positive bacteria, a purple bacteria, a green sulfurbacteria, a green non-sulfur bacteria, a cyanobacteria, a spirochetes, athermatogale, a flavobacteria, bacteroides; a fungal cell, such asSaccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; aninsect cell such as Drosophila and Spodoptera frugiperda; a mammaliancell such as CHO, COS, BHK, HEK 293 or Bowes melanoma, archaebacteria(i.e., Korarchaeota, Thermoproteus, Pyrodictium, Thermococcales,Methanogens, Archaeoglobus, and extreme Halophiles) and others.

F. Polynucleotide Constructs

The use of the term “polynucleotide” is not intended to limit apolynucleotide of the disclosure to a polynucleotide comprising DNA.Those of ordinary skill in the art will recognize that polynucleotidescan comprise ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the disclosure also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

The GLYAT polynucleotides disclosed herein can be provided in expressioncassettes for expression in the plant of interest or any organism ofinterest. The cassette can include 5′ and 3′ regulatory sequencesoperably linked to a GLYAT polynucleotide or active variant or fragmentthereof. “Operably linked” is intended to mean a functional linkagebetween two or more elements. For example, an operable linkage between apolynucleotide of interest and a regulatory sequence (i.e., a promoter)is a functional link that allows for expression of the polynucleotide ofinterest. Operably linked elements may be contiguous or non-contiguous.When used to refer to the joining of two protein coding regions, byoperably linked is intended that the coding regions are in the samereading frame. The cassette may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple expression cassettes.Such an expression cassette is provided with a plurality of restrictionsites and/or recombination sites for insertion of the GLYATpolynucleotide or active variant or fragment thereof to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a GLYAT polynucleotide or active variant or fragmentthereof, and a transcriptional and translational termination region(i.e., termination region) functional in plants. The regulatory regions(i.e., promoters, transcriptional regulatory regions, and translationaltermination regions) and/or the GLYAT polynucleotide or active variantor fragment thereof may be native/analogous to the host cell or to eachother. Alternatively, the regulatory regions and/or the GLYATpolynucleotide of or active variant or fragment thereof may beheterologous to the host cell or to each other.

As used herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

The termination region may be native with the transcriptional initiationregion or active variant or fragment thereof, may be native with theplant host, or may be derived from another source (i.e., foreign orheterologous) to the promoter, the GLYAT polynucleotide or activefragment or variant thereof, the plant host, or any combination thereof.Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus) (Virology 154:9-20), and human immunoglobulin heavy-chain bindingprotein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslatedleader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4)(Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader(TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV)(Lommel et al. (1991) Virology 81:382-385. See also, Della-Cioppa et al.(1987) Plant Physiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used to express the various GLYAT sequencesdisclosed herein, including the native promoter of the polynucleotidesequence of interest. The promoters can be selected based on the desiredoutcome. Such promoters include, for example, constitutive, inducible,tissue-preferred, or other promoters for expression in plants or in anyorganism of interest.

Constitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al.(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, U.S.Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Tissue-preferred promoters can be utilized to target enhanced GLYATexpression within a particular plant tissue. Tissue-preferred promotersinclude those described in Yamamoto et al. (1997) Plant J.12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803;Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al.(1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) PlantPhysiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524;Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994)Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant MolBiol. 23(6): 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Synthetic promoters can be used to express GLYAT sequences orbiologically active variants and fragments thereof.

Alternatively, a plant promoter may be under environmental control. Suchpromoters are referred to here as “inducible” promoters. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include pathogen attack, anaerobic conditions, or the presenceof light. In particular, examples of inducible promoters are the Adh1promoter which is inducible by hypoxia or cold stress, the Hsp70promoter which is inducible by heat stress, and the PPDK promoter whichis inducible by light. Also useful are promoters which are chemicallyinducible.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners; the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides; andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters. See, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257 and thetetracycline-inducible and tetracycline-repressible promoters forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglyphosate, glufosinate ammonium, bromoxynil, sulfonylureas, dicamba,and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference. Theabove list of selectable marker genes is not meant to be limiting. Anyselectable marker gene can be used in the context of the presentdisclosure, including for example, DsRed.

In another aspect, the GLYAT sequences disclosed herein or activevariants or fragments thereof can also be used as a selectable markergene. In this embodiment, the presence of the GLYAT polynucleotide in acell or organism confers upon the cell or organism the detectablephenotypic trait of glyphosate resistance, thereby allowing one toselect for cells or organisms that have been transformed with a gene ofinterest linked to the GLYAT polynucleotide. Thus, for example, theGLYAT polynucleotide can be introduced into a nucleic acid construct,e.g., a vector, thereby allowing for the identification of a host (e.g.,a cell or transgenic plant) containing the nucleic acid construct bygrowing the host in the presence of glyphosate and selecting for theability to survive and/or grow at a rate that is discernibly greaterthan a host lacking the nucleic acid construct would survive or grow. AGLYAT polynucleotide can be used as a selectable marker in a widevariety of hosts that are sensitive to glyphosate, including plants,most bacteria (including E. coli), actinomycetes, yeasts, algae andfungi. One benefit of using herbicide resistance as a marker in plants,as opposed to conventional antibiotic resistance, is that it obviatesthe concern of some members of the public that antibiotic resistancemight escape into the environment.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the GLYAT polypeptide. For example,when large quantities of GLYAT polypeptide or fragments thereof areneeded for commercial production or for induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified can be desirable. Such vectors include, but are not limited to,multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the GLYAT polypeptide coding sequencemay be ligated into the vector in-frame with sequences for theamino-terminal Met and the subsequent 7 residues of beta-galactosidaseso that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster(1989) J Biol Chem 264:5503-5509); pET vectors (Novagen, Madison Wis.);and the like.

Similarly, in the yeast Saccharomyces cerevisiae a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase and PGH may be used for production of the GLYATpolypeptides of the disclosure. For reviews, see Ausubel (supra) andGrant et al. (1987) Methods in Enzymology 153:516-544.

In mammalian host cells, a variety of expression systems, includingviral-based systems, may be utilized. In cases where an adenovirus isused as an expression vector, a coding sequence, e.g., of a GLYATpolypeptide, is optionally ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion of a GLYAT polypeptide codingregion into a nonessential E1 or E3 region of the viral genome willresult in a viable virus capable of expressing a GLYAT in infected hostcells (Logan and Shenk (1984) Proc Natl Acad Sci USA 81:3655-3659). Inaddition, transcription enhancers, such as the rous sarcoma virus (RSV)enhancer, may be used to increase expression in mammalian host cells.

In specific embodiments, the GLYAT polypeptides and active variants andfragments thereof, and polynucleotides encoding the same, furthercomprise a chloroplast transit peptide. As used herein, the term“chloroplast transit peptide” will be abbreviated “CTP” and refers tothe N-terminal portion of a chloroplast precursor protein that directsthe latter into chloroplasts and is subsequently cleaved off by thechloroplast processing protease. When a CTP is operably linked to theN-terminus of a polypeptide, the polypeptide is translocated into thechloroplast. Removal of the CTP from a native protein reduces orabolishes the ability of the native protein from being transported intothe chloroplast. An operably linked chloroplast transit peptide is foundat the N-terminus of the protein to be targeted to the chloroplast andis located upstream and immediately adjacent to the transit peptidecleavage site that separates the transit peptide from the mature proteinto be targeted to the chloroplast.

The term “chloroplast transit peptide cleavage site” refers to a sitebetween two amino acids in a chloroplast-targeting sequence at which thechloroplast processing protease acts. Chloroplast transit peptidestarget the desired protein to the chloroplast and can facilitate theproteins translocation into the organelle. This is accompanied by thecleavage of the transit peptide from the mature polypeptide or proteinat the appropriate transit peptide cleavage site by a chloroplastprocessing protease, native to the chloroplast. Accordingly, achloroplast transit peptide further comprises a suitable cleavage sitefor the correct processing of the pre-protein to the mature polypeptidecontained within the chloroplast.

As used herein, a “heterologous” CTP comprises a transit peptidesequence which is foreign to the polypeptide it is operably linked to.Such heterologous chloroplast transit peptides are known, including butnot limited to those derived from Pisum (JP 1986224990; E00977), carrot(Luo et al. (1997) Plant Mol. Biol., 33 (4), 709-722 (Z33383), Nicotiana(Bowler et al., EP 0359617; A09029), Oryza (de Pater et al. (1990) PlantMol. Biol., 15 (3), 399-406 (X51911), as well as synthetic sequencessuch as those provided in EP 0189707; U.S. Pat. No. 5,728,925; U.S. Pat.No. 5,717,084 (A10396 and A10398). In one embodiment, the heterologouschloroplast transit peptide is from the ribulose-1,5-bisphosphatecarboxylase (Rubisco) small subunit precursor protein isolated from anyplant. The Rubisco small subunit is well characterized from a variety ofplants and the transit peptide from any of them will be suitable for usedisclosed herein. See for example, Physcomitrella (Quatrano et al.,AW599738); Lotus (Poulsen et al., AW428760); Citrullus (J. S. Shin,A1563240); Nicotiana (Appleby et al. (1997) Heredity 79(6), 557-563);alfalfa (Khoudi et al. (1997) Gene, 197(1/2), 343-351); potato andtomato (Fritz et al. (1993) Gene, 137(2), 271-4); wheat (Galili et al.(1991) Theor. Appl. Genet. 81(1), 98-104); and rice (Xie et al. (1987)Sci. Sin., Ser. B (Engl. Ed.), 30(7), 706-19). For example, transitpeptides may be derived from the Rubisco small subunit isolated fromplants including but not limited to, soybean, rapeseed, sunflower,cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice,Arabidopsis, sugar beet, sugar cane, canola, millet, beans, peas, rye,flax, and forage grasses. Preferred for use in the present disclosure isthe Rubisco small subunit precursor protein from, for example,Arabidopsis or tobacco.

G. Stacking Other Traits of Interest

In some embodiments, the GLYAT polynucleotides or active variants andfragments thereof disclosed herein are engineered into a molecularstack. Thus, the various host cells, plants, plant cells and seedsdisclosed herein can further comprise one or more traits of interest,and in more specific embodiments, the host cell, plant, plant part orplant cell is stacked with any combination of polynucleotide sequencesof interest in order to create plants with a desired combination oftraits. As used herein, the term “stacked” includes having the multipletraits present in the same plant or organism of interest. In onenon-limiting example, “stacked traits” comprise a molecular stack wherethe sequences are physically adjacent to each other. A trait, as usedherein, refers to the phenotype derived from a particular sequence orgroups of sequences. In one embodiment, the molecular stack comprises atleast one additional polynucleotide that also confers tolerance to atleast one sequence that confers tolerance to glyphosate by the sameand/or different mechanism and/or at least one additional polynucleotidethat confers tolerance to a second herbicide.

Thus, in one embodiment, the host cells, plants, plant cells or plantpart having the GLYAT polynucleotide or active variants or fragmentsthereof disclosed herein is stacked with at least one other GLYATsequence. Such GLYAT sequence include the GLYAT sequence and variantsand fragment thereof disclosed herein, as well as other GLYAT sequence,which include but are not limited to, the GLYAT sequences set forth inWO02/36782, US Publication 2004/0082770 and WO 2005/012515, U.S. Pat.No. 7,462,481, U.S. Pat. No. 7,405,074, each of which is hereinincorporated by reference.

In some embodiments, the host cells, plants or plant cells having theGLYAT polynucleotides or active variants or fragments thereof may bestacked with other herbicide-tolerance traits to create a transgenicplant of the disclosure with further properties. Thus the mechanism ofglyphosate resistance conferred by GLYAT can be combined with othermodes of glyphosate resistance known in the art. Glyphosate-tolerancetraits which confer tolerance to glyphosate via a different mechanismthan GLYAT include a sequence that encodes a glyphosate oxido-reductaseenzyme as described more fully in U.S. Pat. Nos. 5,776,760 and5,463,175, each of which is incorporated by reference. Other traitsinclude polynucleotides that confer on the plant the capacity to producea higher level or glyphosate insensitive5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), for example, asmore fully described in U.S. Pat. Nos. 6,248,876 B1; 5,627,061;5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642;4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060;4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and U.S. Pat.No. 5,491,288; and international publications WO 97/04103; WO 00/66746;WO 01/66704; WO 00/66747, WO2007064828, WO2006110586, WO2007146765,WO2008002964, US App. Pubs. 2009/0307802, 201/0197499, 2009/0209427, andU.S. Pat. Nos. 8,436,159 and 6,040,497.

The mechanism of glyphosate resistance produced by the GLYAT sequencesdisclosed herein may be combined with other modes of herbicideresistance to provide host cells, plants, plant explants and plant cellsthat are resistant to glyphosate and one or more other herbicides. Forexample, the plant or plant cell or plant part having the GLYAT sequenceor an active variant or fragment thereof is stacked with, for example, asequence which confers tolerance to an ALS inhibitor. As used herein, an“ALS inhibitor-tolerant polypeptide” comprises any polypeptide whichwhen expressed in a plant confers tolerance to at least one ALSinhibitor. A variety of ALS inhibitors are known and include, forexample, sulfonylurea, imidazolinone, triazolopyrimidines,pryimidinyoxy(thio)benzoates, and/or sulfonylaminocarbonyltriazolinoneherbicides. Additional ALS inhibitors are known and are disclosedelsewhere herein. It is known in the art that ALS mutations fall intodifferent classes with regard to tolerance to sulfonylureas,imidazolinones, triazolopyrimidines, and pyrimidinyl(thio)benzoates,including mutations having the following characteristics: (1) broadtolerance to all four of these groups; (2) tolerance to imidazolinonesand pyrimidinyl(thio)benzoates; (3) tolerance to sulfonylureas andtriazolopyrimidines; and (4) tolerance to sulfonylureas andimidazolinones.

Various ALS inhibitor-tolerant polypeptides can be employed. In someembodiments, the ALS inhibitor-tolerant polynucleotides contain at leastone nucleotide mutation resulting in one amino acid change in the ALSpolypeptide. In specific embodiments, the change occurs in one of sevensubstantially conserved regions of acetolactate synthase. See, forexample, Hattori et al. (1995) Molecular Genetics and Genomes246:419-425; Lee et al. (1998) EMBO Journal 7:1241-1248; Mazur et al.(1989) Ann. Rev. Plant Phys. 40:441-470; and U.S. Pat. No. 5,605,011,each of which is incorporated by reference in their entirety. The ALSinhibitor-tolerant polypeptide can be encoded by, for example, the SuRAor SuRB locus of ALS. In specific embodiments, the ALSinhibitor-tolerant polypeptide comprises the C3 ALS mutant, the HRA ALSmutant, the S4 mutant or the S4/HRA mutant or any combination thereof.Different mutations in ALS are known to confer tolerance to differentherbicides and groups (and/or subgroups) of herbicides; see, e.g.,Tranel and Wright (2002) Weed Science 50:700-712. See also, U.S. Pat.Nos. 5,605,011, 5,378,824, 5,141,870, and 5,013,659, each of which isherein incorporated by reference in their entirety. The soybean, maize,and Arabidopsis HRA sequences are disclosed, for example, inWO2007/024782, herein incorporated by reference.

In some embodiments, the ALS inhibitor-tolerant polypeptide conferstolerance to sulfonylurea and/or imidazolinone herbicides. Theproduction of sulfonylurea-tolerant plants and imidazolinone-tolerantplants is described more fully in U.S. Pat. Nos. 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107;5,928,937; and 5,378,824; and international publication WO 96/33270,which are incorporated herein by reference in their entireties for allpurposes. In specific embodiments, the ALS inhibitor-tolerantpolypeptide comprises a sulfonamide-tolerant acetolactate synthase(otherwise known as a sulfonamide-tolerant acetohydroxy acid synthase)or an imidazolinone-tolerant acetolactate synthase (otherwise known asan imidazolinone-tolerant acetohydroxy acid synthase).

In still other embodiments, plants, plant cells, explants and expressioncassettes comprising the GLYAT sequences or active variants andfragments thereof are stacked with a sequence that confers tolerance toan HPPD inhibitor. For example, a P450 sequence could be employed whichprovides tolerance to HPPD-inhibitors by metabolism of the herbicide.Such sequences including, but are not limited to, the NSF1 gene. See, US2007/0214515 and US 2008/0052797, both of which are herein incorporatedby reference in their entirety. In other embodiments, the plants, plantcells, explants and expression cassettes can comprise a GLYAT sequenceor active variant or fragment thereof stacked with an HPPD sequence orvariants and fragments thereof which confers tolerance to an HPPDinhibitor. See, for example, US App. Pub. 2012-0042413 and U.S. Pat.Nos. 6,245,968 B1; 6,268,549; and 6,069,115; and internationalpublication WO 99/23886, U.S. Pat. Nos. 6,245,968 B1; and 6,268,549,each of which is herein incorporated by reference.

In still other embodiments, the plant or plant cell or plant part havingthe GLYAT sequence or an active variants or fragments thereof may bestacked with, for example, aryloxyalkanoate dioxygenase (AAD)polynucleotides (which confer tolerance to 2,4-D and other phenoxy auxinherbicides as well as to aryloxyphenoxypropionate herbicides asdescribed, for example, in WO2005/107437) and dicamba-tolerancepolynucleotides as described, for example, in Herman et al. (2005) J.Biol. Chem. 280: 24759-24767 and U.S. Pat. No. 8,629,328, auxinpolypeptides, acetyl coenzyme A carboxylase (ACCase) polypeptides, andmethyl transferases that provide tolerance to auxin herbicides (e.g,US20130109075, Feng et al.).

In other embodiments, plants, plant cells, explants and expressioncassettes comprising the GLYAT sequences or active variants andfragments thereof are stacked with a sequence that confers tolerance toan inhibitor of Glutamine synthetase (GS). GS appears to be an essentialenzyme necessary for the development and life of most plant cells.Inhibitors of GS are toxic to plant cells. Glufosinate herbicides havebeen developed based on the toxic effect due to the inhibition of GS inplants. These herbicides are non-selective. They inhibit growth of allthe different species of plants present, causing their totaldestruction. The development of plants containing an exogenousphosphinothricin acetyl transferase is described in U.S. Pat. Nos.5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236;5,648,477; 5,646,024; 6,177,616 B1; and 5,879,903, which areincorporated herein by reference in their entireties for all purposes.

Other examples of herbicide-tolerance traits that could be combined withthe plant or plant cell or plant part having the GLYAT sequence or anactive variant or fragment thereof include those conferred bypolynucleotides encoding an exogenous phosphinothricinacetyltransferase, as described in U.S. Pat. Nos. 5,969,213; 5,489,520;5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024;6,177,616; and 5,879,903. Plants containing an exogenousphosphinothricin acetyltransferase can exhibit improved tolerance toglufosinate herbicides, which inhibit the enzyme glutamine synthase.Other examples of herbicide-tolerance traits that could be combined withthe plants or plant cell or plant part having the GLYAT sequence or anactive variant or fragment thereof include those conferred bypolynucleotides conferring altered protoporphyrinogen oxidase (protox)activity, as described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1; and5,767,373; and international publication WO 01/12825. Plants containingsuch polynucleotides can exhibit improved tolerance to any of a varietyof herbicides which target the protox enzyme (also referred to as“protox inhibitors”).

Other examples of herbicide-tolerance traits that could be combined withthe plants or plant cell or plant part having the GLYAT sequence or anactive variant or fragment thereof include those conferring tolerance toat least one herbicide in a plant such as, for example, a maize plant orhorseweed. Herbicide-tolerant weeds are known in the art, as are plantsthat vary in their tolerance to particular herbicides. See, e.g., Greenand Williams (2004) “Correlation of Corn (Zea mays) Inbred Response toNicosulfuron and Mesotrione,” poster presented at the WSSA AnnualMeeting in Kansas City, Mo., Feb. 9-12, 2004; Green (1998) WeedTechnology 12: 474-477; Green and Ulrich (1993) Weed Science 41:508-516. The trait(s) responsible for these tolerances can be combinedby breeding or via other methods with the plants or plant cell or plantpart having the GLYAT sequence or an active variant or fragment thereofto provide a plant of the disclosure as well as methods of use thereof.

In still further embodiments, the GLYAT sequences can be stacked with atleast one polynucleotide encoding a homogentisate solanesyltransferase(HST). See, for example, WO2010023911 herein incorporated by referencein its entirety. In such embodiments, classes of herbicidalcompounds—which act wholly or in part by inhibiting HST can be appliedover the plants having the HTS polypeptide.

The plant or plant cell or plant part having the GLYAT sequence or anactive variant or fragment thereof can also be combined with at leastone other trait to produce plants that further comprise a variety ofdesired trait combinations including, but not limited to, traitsdesirable for animal feed such as high oil content (e.g., U.S. Pat. No.6,232,529); balanced amino acid content (e.g., hordothionins (U.S. Pat.Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409; U.S. Pat. No.5,850,016); barley high lysine (Williamson et al. (1987) Eur. J.Biochem. 165: 99-106; and WO 98/20122) and high methionine proteins(Pedersen et al. (1986) J. Biol. Chem. 261: 6279; Kirihara et al. (1988)Gene 71: 359; and Musumura et al. (1989) Plant Mol. Biol. 12:123));increased digestibility (e.g., modified storage proteins (U.S.application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins(U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)); thedisclosures of which are herein incorporated by reference. Desired traitcombinations also include LLNC (low linolenic acid content; see, e.g.,Dyer et al. (2002) Appl. Microbiol. Biotechnol. 59: 224-230) and OLCH(high oleic acid content; see, e.g., Fernandez-Moya et al. (2005) J.Agric. Food Chem. 53: 5326-5330).

The plant or plant cell or plant part having the GLYAT sequence or anactive variant or fragment thereof can also be combined with otherdesirable traits such as, for example, fumonisim detoxification genes(U.S. Pat. No. 5,792,931), avirulence and disease resistance genes(Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78: 1089), and traits desirable forprocessing or process products such as modified oils (e.g., fatty aciddesaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modifiedstarches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS),starch branching enzymes (SBE), and starch debranching enzymes (SDBE));and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoAreductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847)facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosuresof which are herein incorporated by reference. One could also combineherbicide-tolerant polynucleotides with polynucleotides providingagronomic traits such as male sterility (e.g., see U.S. Pat. No.5,583,210), stalk strength, flowering time, or transformation technologytraits such as cell cycle regulation or gene targeting (e.g., WO99/61619, WO 00/17364, and WO 99/25821); the disclosures of which areherein incorporated by reference.

In still further embodiments, the plant, plant cell, explant or seedcomprises a GLYAT sequence disclosed herein stacked with a sequence thatconfers tolerance to glyphosate via a different mechanism that GLYAT anda sequence that confers tolerance to an additional herbicide. Inspecific embodiments, plants, plant cells, explants or seeds comprise aGLYAT sequence disclosed herein or an active variant or fragment thereofstacked with a sequence encoding a glyphosate tolerant EPSPS and furtherstacked with an sequence encoding an ALS-inhibitor tolerant sequence. Inspecific embodiments, the ALS-inhibitor tolerant sequence is HRA.

In other embodiments, the plant or plant cell or plant part having theGLYAT sequence or an active variant or fragment thereof may be stackedwith any other polynucleotides encoding polypeptides having pesticidaland/or insecticidal activity, such as Bacillus thuringiensis toxicproteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514;5,723,756; 5,593,881; Geiser et al. (1986) Gene 48: 109; Lee et al.(2003) Appl. Environ. Microbiol. 69: 4648-4657 (Vip3A); Galitzky et al.(2001) Acta Crystallogr. D. Biol. Crystallogr. 57: 1101-1109 (Cry3Bb1);and Herman et al. (2004) J. Agric. Food Chem. 52: 2726-2734 (Cry1F)),lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825, pentin(described in U.S. Pat. No. 5,981,722), and the like. The combinationsgenerated can also include multiple copies of any one of thepolynucleotides of interest.

In another embodiment, the plant or plant cell or plant part having theGLYAT sequence or an active variant or fragment thereof can also becombined with a plant disease resistance gene such as but not limited tothe Rcg1 sequence or a biologically active variant or fragment thereof.The Rcg1 sequence is an anthracnose stalk rot resistance gene in corn.See, for example, U.S. patent application Ser. Nos. 11/397,153,11/397,275, and 11/397,247, each of which is herein incorporated byreference.

These stacked combinations can be created by any method including, butnot limited to, breeding plants by any conventional methodology, orgenetic transformation. If the sequences are stacked by geneticallytransforming the plants, the polynucleotide sequences of interest can becombined at any time and in any order. The traits can be introducedsimultaneously in a co-transformation protocol with the polynucleotidesof interest provided by any combination of transformation cassettes. Forexample, if two sequences will be introduced, the two sequences can becontained in separate transformation cassettes (trans) or contained onthe same transformation cassette (cis). Expression of the sequences canbe driven by the same promoter or by different promoters. In certaincases, it may be desirable to introduce a transformation cassette thatwill suppress the expression of the polynucleotide of interest. This maybe combined with any combination of other suppression cassettes oroverexpression cassettes to generate the desired combination of traitsin the plant. It is further recognized that polynucleotide sequences canbe stacked at a desired genomic location using a site-specificrecombination system. See, for example, WO99/25821, WO99/25854,WO99/25840, WO99/25855, and WO99/25853, all of which are hereinincorporated by reference.

Any plant having at GLYAT sequence disclosed herein or an active variantor fragment thereof can be used to make a food or a feed product. Suchmethods comprise obtaining a plant, explant, seed, plant cell, or cellcomprising the GLYAT sequence or active variant or fragment thereof andprocessing the plant, explant, seed, plant cell, or cell to produce afood or feed product.

H. Method of Introducing

Various methods can be used to introduce a sequence of interest into ahost cell, plant or plant part. “Introducing” is intended to meanpresenting to the host cell, plant, plant cell or plant part thepolynucleotide or polypeptide in such a manner that the sequence gainsaccess to the interior of a cell of the plant or organism. The methodsof the disclosure do not depend on a particular method for introducing asequence into an organism or a plant or plant part, only that thepolynucleotide or polypeptides gains access to the interior of at leastone cell of the organism or the plant. Methods for introducingpolynucleotide or polypeptides into various organisms, including plants,are known in the art including, but not limited to, stabletransformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant or organism of interest and is capable of being inherited by theprogeny thereof. “Transient transformation” is intended to mean that apolynucleotide is introduced into the plant or organism of interest anddoes not integrate into the genome of the plant or organism or apolypeptide is introduced into a plant or organism.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), andballistic particle acceleration (see, for example, U.S. Pat. No.4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. Nos. 5,886,244; and,5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation(WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Nat. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783;and, 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize);Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-VanSlogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York),pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the GLYAT sequences or active variants orfragments thereof can be provided to a plant using a variety oftransient transformation methods. Such transient transformation methodsinclude, but are not limited to, the introduction of the GLYAT proteinor active variants and fragments thereof directly into the plant. Suchmethods include, for example, microinjection or particle bombardment.See, for example, Crossway et al. (1986) Mol Gen. Genet. 202:179-185;Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc.Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal ofCell Science 107:775-784, all of which are herein incorporated byreference.

In other embodiments, the GLYAT polynucleotide disclosed herein oractive variants and fragments thereof may be introduced into plants bycontacting plants with a virus or viral nucleic acids. Generally, suchmethods involve incorporating a nucleotide construct of the disclosurewithin a DNA or RNA molecule. It is recognized that the GLYAT sequencemay be initially synthesized as part of a viral polyprotein, which latermay be processed by proteolysis in vivo or in vitro to produce thedesired recombinant protein. Further, it is recognized that promotersdisclosed herein also encompass promoters utilized for transcription byviral RNA polymerases. Methods for introducing polynucleotides intoplants and expressing a protein encoded therein, involving viral DNA orRNA molecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al.(1996) Molecular Biotechnology 5:209-221; herein incorporated byreference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide disclosed herein can be contained in transfercassette flanked by two non-recombinogenic recombination sites. Thetransfer cassette is introduced into a plant having stably incorporatedinto its genome a target site which is flanked by two non-recombinogenicrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome. Other methods to target polynucleotides are set forth inWO 2009/114321 (herein incorporated by reference), which describes“custom” meganucleases produced to modify plant genomes, in particularthe genome of maize. See, also, Gao et al. (2010) Plant Journal1:176-187.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present disclosure provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide disclosedherein, for example, as part of an expression cassette, stablyincorporated into their genome.

Transformed plant cells which are derived by plant transformationtechniques, including those discussed above, can be cultured toregenerate a whole plant which possesses the transformed genotype (i.e.,a GLYAT polynucleotide), and thus the desired phenotype, such asacquired resistance (i.e., tolerance) to glyphosate or a glyphosateanalog. For transformation and regeneration of maize see, Gordon-Kamm etal., The Plant Cell, 2:603-618 (1990). Plant regeneration from culturedprotoplasts is described in Evans et al. (1983) Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp 124-176, MacmillanPublishing Company, New York; and Binding (1985) Regeneration of Plants,Plant Protoplasts pp 21-73, CRC Press, Boca Raton. Regeneration can alsobe obtained from plant callus, explants, organs, or parts thereof. Suchregeneration techniques are described generally in Klee et al. (1987)Ann Rev of Plant Phys 38:467. See also, e.g., Payne and Gamborg.

One of skill will recognize that after the expression cassettecontaining the GLYAT gene is stably incorporated in transgenic plantsand confirmed to be operable, it can be introduced into other plants bysexual crossing. Any of a number of standard breeding techniques can beused, depending upon the species to be crossed.

In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, mature transgenic plants canbe self crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included, provided that theseparts comprise cells comprising the GLYAT nucleic acid. Progeny andvariants, and mutants of the regenerated plants are also included,provided that these parts comprise the introduced nucleic acidsequences.

In one embodiment, a homozygous transgenic plant can be obtained bysexually mating (selfing) a heterozygous transgenic plant that containsa single added heterologous nucleic acid, germinating some of the seedproduced and analyzing the resulting plants produced for altered celldivision relative to a control plant (i.e., native, non-transgenic).Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated.

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These methodsinclude: calcium phosphate precipitation; fusion of the recipient cellswith bacterial protoplasts containing the DNA; treatment of therecipient cells with liposomes containing the DNA; DEAE dextran;electroporation; biolistics; and micro-injection of the DNA directlyinto the cells. The transfected cells are cultured by means well knownin the art. See, Kuchler, R. J., Biochemical Methods in Cell Culture andVirology, Dowden, Hutchinson and Ross, Inc. (1977).

II. Methods of Use

A. Methods for Increasing Expression and/or Activity Level of at LeastOne GLYAT Sequence or an Active Variant or Fragment Therefore in an HostCell of Interest, a Plant or Plant Part

Various methods are provided for the expression of a GLYAT sequence oractive variant or fragment thereof in a host cell of interest. Forexample, the host cell of interest is transformed with the GLYATsequence and the cells are cultured under conditions which allow for theexpression of the GLYAT sequence. In some embodiments, the cells areharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, or other methods,which are well known to those skilled in the art.

As noted, many references are available for the culture and productionof many cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin. See e.g., Sambrook, Ausubel, andBerger (all supra), as well as Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, 3^(rd) Ed., Wiley-Liss, New York and thereferences cited therein; Doyle and Griffiths (1997) Mammalian CellCulture: Essential Techniques John Wiley and Sons, NY; Humason (1979)Animal Tissue Techniques, 4^(th) Ed. W. H. Freeman and Company; andRicciardelli, et al., (1989) In vitro Cell Dev. Biol. 25:1016-1024. Forplant cell culture and regeneration see, Payne et al. (1992) Plant Celland Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York,N.Y.; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and OrganCulture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin, Heidelberg, New York); Jones, ed. (1984) Plant Gene Transferand Expression Protocols, Humana Press, Totowa, N.J.; and PlantMolecular Biology (1993) R. R. D. Croy, ed. Bios Scientific Publishers,Oxford, U.K. ISBN 0 12 198370 6. Cell culture media in general are setforth in Atlas and Parks (eds.) The Handbook of Microbiological Media(1993) CRC Press, Boca Raton, Fla. Additional information for cellculture is found in available commercial literature such as the LifeScience Research Cell Culture Catalogue (1998) from Sigma-Aldrich, Inc.(St Louis, Mo.) (“Sigma-LSRCCC”) and, e.g., The Plant Culture Catalogueand supplement (1997) also from Sigma-Aldrich, Inc. (St Louis, Mo.)(“Sigma-PCCS”).

A method for increasing the activity of a GLYAT polypeptide disclosedherein or an active variant or fragment thereof in a plant, plant cell,plant part, explant, and/or seed is provided. In further embodiments,the activity of the GLYAT polypeptide is increased in a plant or plantpart by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,200%, 500%, 1000%, 5000%, or 10,000% relative to an appropriate controlplant, plant part, or cell. In still other embodiments, the activitylevel of the GLYAT polypeptide in the plant or plant part is increasedby 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 fold or more relative to anappropriate control plant, plant part, or cell. Such an increase in theactivity of the GLYAT polypeptide in the cell can be achieved in avariety of ways including, for example, by the expression of multiplecopies of one or more GLYAT polypeptide, by employing a promoter todrive higher levels of expression of the sequence, or by employing aGLYAT sequence having an increased level of activity.

In specific embodiments, the polypeptide or the GLYAT polynucleotide oractive variant or fragment thereof is introduced into the plant, plantcell, explant or plant part. Subsequently, a plant cell having anintroduced sequence disclosed herein is selected using methods known tothose of skill in the art such as, but not limited to, Southern blotanalysis, DNA sequencing, PCR analysis, or phenotypic analysis. A plantor plant part altered or modified by the foregoing embodiments is grownunder plant forming conditions for a time sufficient to modulate thetemporal or spatial expression of polypeptides disclosed herein in theplant. Plant forming conditions are well known in the art and discussedbriefly elsewhere herein.

In one embodiment, a method of producing a glyphosate tolerant plantcell is provided and comprises transforming a plant cell with thepolynucleotide encoding a GLYAT polypeptide or active variant orfragment thereof. In specific embodiments, the method further comprisesselecting a plant cell which is resistant or tolerant to a glyphosate bygrowing the plant cells in a sufficient concentration of glyphosate,such that the herbicide kills the plant cells which do not comprise theGLYAT polypeptide of interest.

B. Method of Producing Crops and Controlling Weeds

Methods for controlling weeds in an area of cultivation, preventing thedevelopment or the appearance of herbicide resistant weeds in an area ofcultivation, producing a crop, and increasing crop safety are provided.The term “controlling,” and derivations thereof, for example, as in“controlling weeds” refers to one or more of inhibiting the growth,germination, reproduction, and/or proliferation of; and/or killing,removing, destroying, or otherwise diminishing the occurrence and/oractivity of a weed.

As used herein, an “area of cultivation” comprises any region in whichone desires to grow a plant. Such areas of cultivations include, but arenot limited to, a field in which a plant is cultivated (such as a cropfield, a sod field, a tree field, a managed forest, a field forculturing fruits and vegetables, etc), a greenhouse, a growth chamber,etc.

As used herein, by “selectively controlled” it is intended that themajority of weeds in an area of cultivation are significantly damaged orkilled, while if crop plants are also present in the field, the majorityof the crop plants are not significantly damaged. Thus, a method isconsidered to selectively control weeds when at least 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or more of the weeds are significantlydamaged or killed, while if crop plants are also present in the field,less than 10%, 5%, or 1% of the crop plants are significantly damaged orkilled.

Methods provided comprise planting the area of cultivation with a planthaving a GLYAT sequence or active variant or fragment thereof disclosedherein or transgenic seed derived therefrom, and in specificembodiments, applying to the crop, seed, weed or area of cultivationthereof an effective amount of a herbicide of interest. It is recognizedthat the herbicide can be applied before or after the crop is planted inthe area of cultivation. Such herbicide applications can include anapplication of glyphosate.

Accordingly, the term “glyphosate” should be considered to include anyherbicidally effective form of N-phosphonomethylglycine (including anysalt thereof) and other forms which result in the production of theglyphosate anion in planta.

In specific methods, glyphosate is applied to the plants having theGLYAT sequence or active variant or fragment thereof or their area ofcultivation. In specific embodiments, the glyphosate is in the form of asalt, such as, ammonium, isopropylammonium, potassium, sodium (includingsesquisodium) or trimesium (alternatively named sulfosate). In stillfurther embodiments, a mixture of a synergistically effective amount ofa combination of glyphosate and an ALS inhibitor (such as asulfonylurea) is applied to the plants or their area of cultivation.

Generally, the effective amount of herbicide applied to the field issufficient to selectively control the weeds without significantlyaffecting the crop. It is important to note that it is not necessary forthe crop to be totally insensitive to the herbicide, so long as thebenefit derived from the inhibition of weeds outweighs any negativeimpact of the glyphosate or glyphosate analog on the crop or crop plant.

“Weed” as used herein refers to a plant which is not desirable in aparticular area. Conversely, a “crop plant” as used herein refers to aplant which is desired in a particular area, such as, for example, amaize or soy plant. Thus, in some embodiments, a weed is a non-cropplant or a non-crop species, while in some embodiments, a weed is a cropspecies which is sought to be eliminated from a particular area, suchas, for example, an inferior and/or non-transgenic soy plant in a fieldplanted with a plant having the GLYAT sequence disclosed herein or anactive variant or fragment thereof.

Accordingly, the current disclosure provides methods for selectivelycontrolling weeds in a field containing a crop that involve planting thefield with crop seeds or plants which are glyphosate-tolerant as aresult of being transformed with a gene encoding aglyphosate-N-acetyltransferase disclosed herein or an active variant orfragment thereof, and applying to the crop and weeds in the field asufficient amount of glyphosate to control the weeds withoutsignificantly affecting the crop.

Further provided are methods for controlling weeds in a field andpreventing the emergence of glyphosate resistant weeds in a fieldcontaining a crop which involve planting the field with crop seeds orplants that are glyphosate tolerant as a result of being transformedwith a gene encoding a glyphosate-N-acetyltransferase and a geneencoding a polypeptide imparting glyphosate tolerance by anothermechanism, such as, a glyphosate-tolerant5-enolpyruvylshikimate-3-phosphate synthase and/or a glyphosate-tolerantglyphosate oxido-reductase and applying to the crop and the weeds in thefield a sufficient amount of glyphosate to control the weeds withoutsignificantly affecting the crop. Various plants that can be used inthis method are discussed in detail elsewhere herein.

In further embodiments, the current disclosure provides methods forcontrolling weeds in a field and preventing the emergence of herbicideresistant weeds in a field containing a crop which involve planting thefield with crop seeds or plants that are glyphosate tolerant as a resultof being transformed with a gene encoding aglyphosate-N-acetyltransferase, a gene encoding a polypeptide impartingglyphosate tolerance by another mechanism, such as, aglyphosate-insensitive 5-enolpyruvylshikimate-3-phosphate synthaseand/or a glyphosate oxido-reductase and a gene encoding a polypeptideimparting tolerance to an additional herbicide, such as, a mutatedhydroxyphenylpyruvatedioxygenase, a sulfonylurea-tolerant acetolactatesynthase, a sulfonylurea-tolerant acetohydroxy acid synthase, asulfonamide-tolerant acetolactate synthase, a sulfonamide-tolerantacetohydroxy acid synthase, an imidazolinone-tolerant acetolactatesynthase, an imidazolinone-tolerant acetohydroxy acid synthase, aphosphinothricin acetyl transferase and a mutated protoporphyrinogenoxidase and applying to the crop and the weeds in the field a sufficientamount of glyphosate and an additional herbicide, such as, ahydroxyphenylpyruvatedioxygenase inhibitor, sulfonamide, imidazolinone,bialaphos, phosphinothricin, azafenidin, butafenacil, sulfosate,glufosinate, and a protox inhibitor to control the weeds withoutsignificantly affecting the crop. Various plants and seeds that can beused in this method are discussed in detail elsewhere herein.

Further provided are methods for controlling weeds in a field andpreventing the emergence of herbicide resistant weeds in a fieldcontaining a crop which involve planting the field with crop seeds orplants that are glyphosate tolerant as a result of being transformedwith a gene encoding a glyphosate-N-acetyltransferase and a geneencoding a polypeptide imparting tolerance to an additional herbicide,such as, a mutated hydroxyphenylpyruvatedioxygenase, asulfonamide-tolerant acetolactate synthase, a sulfonamide-tolerantacetohydroxy acid synthase, an imidazolinone-tolerant acetolactatesynthase, an imidazolinone-tolerant acetohydroxy acid synthase, aphosphinothricin acetyl transferase and a mutated protoporphyrinogenoxidase and applying to the crop and the weeds in the field a sufficientamount of glyphosate and an additional herbicide, such as, ahydroxyphenylpyruvatedioxygenase inhibitor, sulfonamide, imidazolinone,bialaphos, phosphinothricin, azafenidin, butafenacil, sulfosate,glufosinate, and a protox inhibitor to control the weeds withoutsignificantly affecting the crop. Various plants and seeds that can beused in this method are discussed in detail elsewhere herein.

Further provided is a method for producing a crop by growing a cropplant that is tolerant to glyphosate as a result of being transformedwith a GLYAT polynucleotide or active variant or fragment thereofdisclosed herein, under conditions such that the crop plant produces acrop, and harvesting the crop. Preferably, the glyphosate is applied tothe plant, or in the vicinity of the plant, at a concentration effectiveto control weeds without preventing the transgenic crop plant fromgrowing and producing the crop. The application of the glyphosate can bebefore planting, or at any time after planting up to and including thetime of harvest. Glyphosate can be applied once or multiple times. Thetiming of glyphosate application, amount applied, mode of application,and other parameters will vary based upon the specific nature of thecrop plant and the growing environment, and can be readily determined byone of skill in the art. A crop produced by this method is alsoprovided.

Further provided are methods for the propagation of a plant containing aGLYAT polypeptide or active variant or fragment thereof. The plant canbe, for example, a monocot or a dicot. In one aspect, propagationentails crossing a plant containing a GLYAT polynucleotide transgenewith a second plant, such that at least some progeny of the crossdisplay glyphosate tolerance.

The methods herein further allow for the development of herbicideapplications to be used with the plants having the GLYAT sequence oractive variants or fragments thereof. In such methods, the environmentalconditions in an area of cultivation are evaluated. Environmentalconditions that can be evaluated include, but are not limited to, groundand surface water pollution concerns, intended use of the crop, croptolerance, soil residuals, weeds present in area of cultivation, soiltexture, pH of soil, amount of organic matter in soil, applicationequipment, and tillage practices. Upon the evaluation of theenvironmental conditions, an effective amount of a combination ofherbicides can be applied to the crop, crop part, and seed of the cropor area of cultivation.

Any herbicide or combination of herbicides can be applied to the planthaving the GLYAT sequence or active variant or fragment thereofdisclosed herein or transgenic seed derived there from, crop part, orthe area of cultivation containing the crop plant. By “treated with acombination of” or “applying a combination of” herbicides to a crop,area of cultivation or field” it is intended that a particular field,crop or weed is treated with each of the herbicides and/or chemicalsindicated to be part of the combination so that a desired effect isachieved, i.e., so that weeds are selectively controlled while the cropis not significantly damaged. The application of each herbicide and/orchemical may be simultaneous or the applications may be at differenttimes (sequential), so long as the desired effect is achieved.Furthermore, the application can occur prior to the planting of thecrop.

Classifications of herbicides (i.e., the grouping of herbicides intoclasses and subclasses) are well-known in the art and includeclassifications by HRAC (Herbicide Resistance Action Committee) and WSSA(the Weed Science Society of America) (see also, Retzinger andMallory-Smith (1997) Weed Technology 11: 384-393). An abbreviatedversion of the HRAC classification (with notes regarding thecorresponding WSSA group) is set forth below in Table 1.

Herbicides can be classified by their mode of action and/or site ofaction and can also be classified by the time at which they are applied(e.g., preemergent or postemergent), by the method of application (e.g.,foliar application or soil application), or by how they are taken up byor affect the plant or by their structure. “Mode of action” generallyrefers to the metabolic or physiological process within the plant thatthe herbicide inhibits or otherwise impairs, whereas “site of action”generally refers to the physical location or biochemical site within theplant where the herbicide acts or directly interacts. Herbicides can beclassified in various ways, including by mode of action and/or site ofaction (see, e.g., Table 1).

Often, an herbicide-tolerance gene that confers tolerance to aparticular herbicide or other chemical on a plant expressing it willalso confer tolerance to other herbicides or chemicals in the same classor subclass, for example, a class or subclass set forth in Table 1.Thus, in some embodiments, a transgenic plant is tolerant to more thanone herbicide or chemical in the same class or subclass, such as, forexample, an HPPD inhibitor, glyphosate, an ALS chemistry, an inhibitorof PPO, a sulfonylurea, and/or a synthetic auxin.

Typically, the plants of the present disclosure can tolerate treatmentwith different types of herbicides (i.e., herbicides having differentmodes of action and/or different sites of action) thereby permittingimproved weed management strategies that are recommended in order toreduce the incidence and prevalence of herbicide-tolerant weeds.

TABLE 1 Abbreviated version of HRAC Herbicide Classification I. ALSInhibitors (WSSA Group 2) A. Sulfonylureas 1. Azimsulfuron 2.Chlorimuron-ethyl 3. Metsulfuron-methyl 4. Nicosulfuron 5. Rimsulfuron6. Sulfometuron-methyl 7. Thifensulfuron-methyl 8. Tribenuron-methyl 9.Amidosulfuron 10. Bensulfuron-methyl 11. Chlorsulfuron 12. Cinosulfuron13. Cyclosulfamuron 14. Ethametsulfuron-methyl 15. Ethoxysulfuron 16.Flazasulfuron 17. Flupyrsulfuron-methyl 18. Foramsulfuron 19.Imazosulfuron 20. Iodosulfuron-methyl 21. Mesosulfuron-methyl 22.Oxasulfuron 23. Primisulfuron-methyl 24. Prosulfuron 25.Pyrazosulfuron-ethyl 26. Sulfosulfuron 27. Triasulfuron 28.Trifloxysulfuron 29. Triflusulfuron-methyl 30. Tritosulfuron 31.Halosulfuron-methyl 32. Flucetosulfuron B.Sulfonylaminocarbonyltriazolinones 1. Flucarbazone 2. Procarbazone C.Triazolopyrimidines 1. Cloransulam-methyl 2. Flumetsulam 3. Diclosulam4. Florasulam 5. Metosulam 6. Penoxsulam 7. Pyroxsulam D.Pyrimidinyloxy(thio)benzoates 1. Bispyribac 2. Pyriftalid 3.Pyribenzoxim 4. Pyrithiobac 5. Pyriminobac-methyl E. Imidazolinones 1.Imazapyr 2. Imazethapyr 3. Imazaquin 4. Imazapic 5.Imazamethabenz-methyl 6. Imazamox II. Other Herbicides--ActiveIngredients/ Additional Modes of Action A. Inhibitors of Acetyl CoAcarboxylase (ACCase) (WSSA Group 1) 1. Aryloxyphenoxypropionates(‘FOPs’) a. Quizalofop-P-ethyl b. Diclofop-methyl c.Clodinafop-propargyl d. Fenoxaprop-P-ethyl e. Fluazifop-P-butyl f.Propaquizafop g. Haloxyfop-P-methyl h. Cyhalofop-butyl i.Quizalofop-P-ethyl 2. Cyclohexanediones (‘DIMs’) a. Alloxydim b.Butroxydim c. Clethodim d. Cycloxydim e. Sethoxydim f. Tepraloxydim g.Tralkoxydim B. Inhibitors of Photosystem II - HRAC Group C1/WSSA Group5 1. Triazines a. Ametryne b. Atrazine c. Cyanazine d. Desmetryne e.Dimethametryne f. Prometon g. Prometryne h. Propazine i. Simazine j.Simetryne k. Terbumeton I. Terbuthylazine m. Terbutryne n. Trietazine 2.Triazinones a. Hexazinone b. Metribuzin c. Metamitron 3. Triazolinone a.Amicarbazone 4. Uracils a. Bromacil b. Lenacil c. Terbacil 5.Pyridazinones a. Pyrazon 6. Phenyl carbamates a. Desmedipham b.Phenmedipham C. Inhibitors of Photosystem II--HRAC Group C2/WSSA Group7 1. Ureas a. Fluometuron b. Linuron c. Chlorobromuron d. Chlorotolurone. Chloroxuron f. Dimefuron g. Diuron h. Ethidimuron i. Fenuron j.Isoproturon k. Isouron I. Methabenzthiazuron m. Metobromuron n.Metoxuron o. Monolinuron p. Neburon q. Siduron r. Tebuthiuron 2. Amidesa. Propanil b. Pentanochlor D. Inhibitors of Photosystem II--HRAC GroupC3/WSSA Group 6 1. Nitriles a. Bromofenoxim b. Bromoxynil c. Ioxynil 2.Benzothiadiazinone (Bentazon) a. Bentazon 3. Phenylpyridazines a.Pyridate b. Pyridafol E. Photosystem-I- electron diversion(Bipyridyliums) (WSSA Group 22) 1. Diquat 2. Paraquat F. Inhibitors ofPPO (protoporphyrinogen oxidase) (WSSA Group 14) 1. Diphenylethers a.Acifluorfen-Na b. Bifenox c. Chlomethoxyfen d. Fluoroglycofen-ethyl e.Fomesafen f. Halosafen g. Lactofen h. Oxyfluorfen 2. Phenylpyrazoles a.Fluazolate b. Pyraflufen-ethyl 3. N-phenylphthalimides a. Cinidon-ethylb. Flumioxazin c. Flumiclorac-pentyl 4. Thiadiazoles a.Fluthiacet-methyl b. Thidiazimin 5. Oxadiazoles a. Oxadiazon b.Oxadiargyl 6. Triazolinones a. Carfentrazone-ethyl b. Sulfentrazone 7.Oxazolidinediones a. Pentoxazone 8. Pyrimidindiones a. Benzfendizone b.Butafenicil 9. Others a. Pyrazogyl b. Profluazol G. Bleaching:Inhibition of carotenoid biosynthesis at the phytoene desaturase step(PDS) (WSSA Group 12) 1. Pyridazinones a. Norflurazon 2.Pyridinecarboxamides a. Diflufenican b. Picolinafen 3. Others a.Beflubutamid b. Fluridone c. Flurochloridone d. Flurtamone H. Bleaching:Inhibition of 4- hydroxphenyl-pyruvate-dioxygenase (4- HPPD) (WSSA Group28) 1. Triketones a. Mesotrione b. Sulcotrione c. topramezone d.tembotrione 2. Isoxazoles a. Pyrasulfotole b. Isoxaflutole 3. Pyrazolesa. Benzofenap b. Pyrazoxyfen c. Pyrazolynate 4. Others a. BenzobicyclonI. Bleaching: Inhibition of carotenoid biosynthesis (unknown target)(WSSA Group 11 and 13) 1. Triazoles (WSSA Group 11) a. Amitrole 2.Isoxazolidinones (WSSA Group 13) a. Clomazone 3. Ureas a. Fluometuron 3.Diphenylether a. Aclonifen J. Inhibition of EPSP Synthase 1. Glycines(WSSA Group 9) a. Glyphosate b. Sulfosate K. Inhibition of glutaminesynthetase 1. Phosphinic Acids a. Glufosinate-ammonium b. Bialaphos L.Inhibition of DHP (dihydropteroate) synthase (WSSA Group 18) 1Carbamates a. Asulam M. Microtubule Assembly Inhibition (WSSA Group3) 1. Dinitroanilines a. Benfluralin b. Butralin c. Dinitramine d.Ethalfluralin e. Oryzalin f. Pendimethalin g. Trifluralin 2.Phosphoroamidates a. Amiprophos-methyl b. Butamiphos 3. Pyridines a.Dithiopyr b. Thiazopyr 4. Benzamides a. Pronamide b. Tebutam 5.Benzenedicarboxylic acids a. Chlorthal-dimethyl N. Inhibition ofmitosis/microtubule organization WSSA Group 23) 1. Carbamates a.Chlorpropham b. Propham c. Carbetamide O. Inhibition of cell division(Inhibition of very long chain fatty acids as proposed mechanism; WSSAGroup 15) 1. Chloroacetamides a. Acetochlor b. Alachlor c. Butachlor d.Dimethachlor e. Dimethanamid f. Metazachlor g. Metolachlor h. Pethoxamidi. Pretilachlor j. Propachlor k. Propisochlor l. Thenylchlor 2.Acetamides a. Diphenamid b. Napropamide c. Naproanilide 3. Oxyacetamidesa. Flufenacet b. Mefenacet 4. Tetrazolinones a. Fentrazamide 5. Othersa. Anilofos b. Cafenstrole c. Indanofan d. Piperophos P. Inhibition ofcell wall (cellulose) synthesis 1. Nitriles (WSSA Group 20) a.Dichlobenil b. Chlorthiamid 2. Benzamides (isoxaben (WSSA Group 21)) a.Isoxaben 3. Triazolocarboxamides (flupoxam) a. Flupoxam Q. Uncoupling(membrane disruption): (WSSA Group 24) 1. Dinitrophenols a. DNOC b.Dinoseb c. Dinoterb R. Inhibition of Lipid Synthesis by other than ACCinhibition 1. Thiocarbamates (WSSA Group 8) a. Butylate b. Cycloate c.Dimepiperate d. EPTC e. Esprocarb f. Molinate g. Orbencarb h. Pebulatei. Prosulfocarb j. Benthiocarb k. Tiocarbazil l. Triallate m. Vernolate2. Phosphorodithioates a. Bensulide 3. Benzofurans a. Benfuresate b.Ethofumesate 4. Halogenated alkanoic acids (WSSA Group 26) a. TCA b.Dalapon c. Flupropanate S. Synthetic auxins (IAA-like) (WSSA Group 4) 1.Phenoxycarboxylic acids a. Clomeprop b. 2,4-D c. Mecoprop 2. Benzoicacids a. Dicamba b. Chloramben c. TBA 3. Pyridine carboxylic acids a.Clopyralid b. Fluroxypyr c. Picloram d. Tricyclopyr 4. Quinolinecarboxylic acids a. Quinclorac b. Quinmerac 5. Others (benazolin-ethyl)a. Benazolin-ethyl T. Inhibition of Auxin Transport 1. Phthalamates;semicarbazones (WSSA Group 19) a. Naptalam b. Diflufenzopyr-Na U. OtherMechanism of Action 1. Arylaminopropionic acids a. Flamprop-M-methyl/-isopropyl 2. Pyrazolium a. Difenzoquat 3. Organoarsenicals a. DSMA b.MSMA 4. Others a. Bromobutide b. Cinmethylin c. Cumyluron d. Dazomet e.Daimuron-methyl f. Dimuron g. Etobenzanid h. Fosamine i. Metam j.Oxaziclomefone k. Oleic acid l. Pelargonic acid m. Pyributicarb

In still further methods, glyphosate can be applied alone or incombination with another herbicide of interest and can be applied to theplants having the GLYAT sequence as disclosed herein or their area ofcultivation.

Additional herbicide treatment that can be applied over the plants orseeds having the GLYAT polypeptides or active variants and fragmentsthereof include, but are not limited to: acetochlor, acifluorfen and itssodium salt, aclonifen, acrolein (2-propenal), alachlor, alloxydim,ametryn, amicarbazone, amidosulfuron, aminopyralid, amitrole, ammoniumsulfamate, anilofos, asulam, atrazine, azimsulfuron, beflubutamid,benazolin, benazolin-ethyl, bencarbazone, benfluralin, benfuresate,bensulfuron-methyl, bensulide, bentazone, benzobicyclon, benzofenap,bifenox, bilanafos, bispyribac and its sodium salt, bromacil,bromobutide, bromofenoxim, bromoxynil, bromoxynil octanoate, butachlor,butafenacil, butamifos, butralin, butroxydim, butylate, cafenstrole,carbetamide, carfentrazone-ethyl, catechin, chlomethoxyfen, chloramben,chlorbromuron, chlorflurenol-methyl, chloridazon, chlorimuron-ethyl,chlorotoluron, chlorpropham, chlorsulfuron, chlorthal-dimethyl,chlorthiamid, cinidon-ethyl, cinmethylin, cinosulfuron, clethodim,clodinafop-propargyl, clomazone, clomeprop, clopyralid,clopyralid-olamine, cloransulam-methyl, CUH-35 (2-methoxyethyl2-[[[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxy]phenyl](3-fluorobenzoyl)amino]carbonyl]-1-cyclohexene-1-carboxylate),cumyluron, cyanazine, cycloate, cyclosulfamuron, cycloxydim,cyhalofop-butyl, 2,4-D and its butotyl, butyl, isoctyl and isopropylesters and its dimethylammonium, diolamine and trolamine salts,daimuron, dalapon, dalapon-sodium, dazomet, 2,4-DB and itsdimethylammonium, potassium and sodium salts, desmedipham, desmetryn,dicamba and its diglycolammonium, dimethylammonium, potassium and sodiumsalts, dichlobenil, dichlorprop, diclofop-methyl, diclosulam,difenzoquat metilsulfate, diflufenican, diflufenzopyr, dimefuron,dimepiperate, dimethachlor, dimethametryn, dimethenamid, dimethenamid-P,dimethipin, dimethylarsinic acid and its sodium salt, dinitramine,dinoterb, diphenamid, diquat dibromide, dithiopyr, diuron, DNOC,endothal, EPTC, esprocarb, ethalfluralin, ethametsulfuron-methyl,ethofumesate, ethoxyfen, ethoxysulfuron, etobenzanid, fenoxaprop-ethyl,fenoxaprop-P-ethyl, fentrazamide, fenuron, fenuron-TCA, flamprop-methyl,flamprop-M-isopropyl, flamprop-M-methyl, flazasulfuron, florasulam,fluazifop-butyl, fluazifop-P-butyl, flucarbazone, flucetosulfuron,fluchloralin, flufenacet, flufenpyr, flufenpyr-ethyl, flumetsulam,flumiclorac-pentyl, flumioxazin, fluometuron, fluoroglycofen-ethyl,flupyrsulfuron-methyl and its sodium salt, flurenol, flurenol-butyl,fluridone, flurochloridone, fluroxypyr, flurtamone, fluthiacet-methyl,fomesafen, foramsulfuron, fosamine-ammonium, glufosinate,glufosinate-ammonium, glyphosate and its salts such as ammonium,isopropylammonium, potassium, sodium (including sesquisodium) andtrimesium (alternatively named sulfosate) (See, WO2007/024782, hereinincorporated by reference), halosulfuron-methyl, haloxyfop-etotyl,haloxyfop-methyl, hexazinone, HOK-201(N-(2,4-difluorophenyl)-1,5-dihydro-N-(1-methylethyl)-5-oxo-1-[(tetrahydro-2H-pyran-2-yl)methyl]-4H-1,2,4-triazole-4-carboxamide),imazamethabenz-methyl, imazamox, imazapic, imazapyr, imazaquin,imazaquin-ammonium, imazethapyr, imazethapyr-ammonium, imazosulfuron,indanofan, iodosulfuron-methyl, ioxynil, ioxynil octanoate,ioxynil-sodium, isoproturon, isouron, isoxaben, isoxaflutole,pyrasulfotole, lactofen, lenacil, linuron, maleic hydrazide, MCPA andits salts (e.g., MCPA-dimethylammonium, MCPA-potassium and MCPA-sodium,esters (e.g., MCPA-2-ethylhexyl, MCPA-butotyl) and thioesters (e.g.,MCPA-thioethyl), MCPB and its salts (e.g., MCPB-sodium) and esters(e.g., MCPB-ethyl), mecoprop, mecoprop-P, mefenacet, mefluidide,mesosulfuron-methyl, mesotrione, metam-sodium, metamifop, metamitron,metazachlor, methabenzthiazuron, methylarsonic acid and its calcium,monoammonium, monosodium and disodium salts, methyldymron, metobenzuron,metobromuron, metolachlor, S-metholachlor, metosulam, metoxuron,metribuzin, metsulfuron-methyl, molinate, monolinuron, naproanilide,napropamide, naptalam, neburon, nicosulfuron, norflurazon, orbencarb,oryzalin, oxadiargyl, oxadiazon, oxasulfuron, oxaziclomefone,oxyfluorfen, paraquat dichloride, pebulate, pelargonic acid,pendimethalin, penoxsulam, pentanochlor, pentoxazone, perfluidone,pethoxyamid, phenmedipham, picloram, picloram-potassium, picolinafen,pinoxaden, piperofos, pretilachlor, prim isulfuron-methyl, prodiamine,profoxydim, prometon, prometryn, propachlor, propanil, propaquizafop,propazine, propham, propisochlor, propoxycarbazone, propyzamide,prosulfocarb, prosulfuron, pyraclonil, pyraflufen-ethyl, pyrasulfotole,pyrazogyl, pyrazolynate, pyrazoxyfen, pyrazosulfuron-ethyl,pyribenzoxim, pyributicarb, pyridate, pyriftalid, pyriminobac-methyl,pyrimisulfan, pyrithiobac, pyrithiobac-sodium, pyroxsulam, quinclorac,quinmerac, quinoclamine, quizalofop-ethyl, quizalofop-P-ethyl,quizalofop-P-tefuryl, rimsulfuron, sethoxydim, siduron, simazine,simetryn, sulcotrione, sulfentrazone, sulfometuron-methyl,sulfosulfuron, 2,3,6-TBA, TCA, TCA-sodium, tebutam, tebuthiuron,tefuryltrione, tembotrione, tepraloxydim, terbacil, terbumeton,terbuthylazine, terbutryn, thenylchlor, thiazopyr, thiencarbazone,thifensulfuron-methyl, thiobencarb, tiocarbazil, topramezone,tralkoxydim, tri-allate, triasulfuron, triaziflam, tribenuron-methyl,triclopyr, triclopyr-butotyl, triclopyr-triethylammonium, tridiphane,trietazine, trifloxysulfuron, trifluralin, triflusulfuron-methyl,tritosulfuron and vernolate.

Additional herbicides include those that are applied over plants havinghomogentisate solanesyltransferase (HST) polypeptide such as thosedescribed in WO2010029311(A2), herein incorporated by reference it itsentirety.

Other herbicides that can be used when an HPPD inhibitor tolerantsequence is present in the plant (in addition to a GLYAT sequencedisclosed herein) include, but are not limited to, triketones (such as,mesotrione, sulcotrione, topremezone, and tembotrione) includingagriculturally suitable salts (e.g., sodium salts) thereof; isoxazoles(such as, pyrasulfotole and isoxaflutole) including agriculturallysuitable salts (e.g., sodium salts) thereof; pyrazoles (such as,benzofenap, pyrazoxyfen, and pyrazolynate) including agriculturallysuitable salts (e.g., sodium salts) thereof; and benzobicyclon,including agriculturally suitable salts (e.g., sodium salts) thereof.See, WO2005/053407. In specific embodiments, a combination of two ormore HPPD inhibitors is applied.

In some embodiments, topical application of polynucleotides (e.g.,dsRNA, ssRNA, constructs expressing hairpin dsRNA, RNA moleculescomprising synthetic nucleotides) to control weeds in crop growing fieldinclude crop plants expressing GLYAT sequences disclosed herein. Suchweed control options include RNA suppression techniques that target oneor more specific endogenous transcripts present in weeds, but do notsubstantially impact the crop plants that are tolerant to glyphosate.

Other suitable herbicides and agricultural chemicals are known in theart, such as, for example, those described in WO 2005/041654. Otherherbicides also include bioherbicides such as Alternaria destruensSimmons, Colletotrichum gloeosporiodes (Penz.) Penz. & Sacc., Drechsieramonoceras (MTB-951), Myrothecium verrucaria (Albertini & Schweinitz)Ditmar: Fries, Phytophthora palmivora (Butl.) Butl. and Pucciniathlaspeos Schub. Combinations of various herbicides can result in agreater-than-additive (i.e., synergistic) effect on weeds and/or aless-than-additive effect (i.e. safening) on crops or other desirableplants. In certain instances, combinations of glyphosate with otherherbicides having a similar spectrum of control but a different mode ofaction will be particularly advantageous for preventing the developmentof resistant weeds.

The time at which a herbicide is applied to an area of interest (and anyplants therein) may be important in optimizing weed control. The time atwhich a herbicide is applied may be determined with reference to thesize of plants and/or the stage of growth and/or development of plantsin the area of interest, e.g., crop plants or weeds growing in the area.

Ranges of the effective amounts of herbicides can be found, for example,in various publications from University Extension services. See, forexample, Bernards et al. (2006) Guide for Weed Management in Nebraska(www.ianrpubs.url.edu/sendlt/ec130); Regher et al. (2005) Chemical WeedControl for Fields Crops, Pastures, Rangeland, and Noncropland, KansasState University Agricultural Extension Station and Corporate ExtensionService; Zollinger et al. (2006) North Dakota Weed Control Guide, NorthDakota Extension Service, and the Iowa State University Extension atwww.weeds.iastate.edu, each of which is herein incorporated byreference.

Many plant species can be controlled (i.e., killed or damaged) by theherbicides described herein. Accordingly, the methods of the disclosureare useful in controlling these plant species where they are undesirable(i.e., where they are weeds). These plant species include crop plants aswell as species commonly considered weeds, including but not limited tospecies such as: blackgrass (Alopecurus myosuroides), giant foxtail(Setaria faberi), large crabgrass (Digitaria sanguinalis), Surinam grass(Brachiaria decumbens), wild oat (Avena fatua), common cocklebur(Xanthium pensylvanicum), common lambsquarters (Chenopodium album),morning glory (Ipomoea coccinea), pigweed (Amaranthus spp.), velvetleaf(Abutilion theophrasti), common barnyardgrass (Echinochloa crusgalli),bermudagrass (Cynodon dactylon), downy brome (Bromus tectorum),goosegrass (Eleusine indica), green foxtail (Setaria viridis), Italianryegrass (Lolium multiflorum), Johnsongrass (Sorghum halepense), lessercanarygrass (Phalaris minor), windgrass (Apera spica-venti), woolycupgrass (Erichloa villosa), yellow nutsedge (Cyperus esculentus),common chickweed (Stellaria media), common ragweed (Ambrosiaartemisiifolia), Kochia scoparia, horseweed (Conyza canadensis), rigidryegrass (Lolium rigidum), goosegrass (Eleucine indica), hairy fleabane(Conyza bonariensis), buckhorn plantain (Plantago lanceolata), tropicalspiderwort (Commelina benghalensis), field bindweed (Convolvulusarvensis), purple nutsedge (Cyperus rotundus), redvine (Brunnichiaovata), hemp sesbania (Sesbania exaltata), sicklepod (Sennaobtusifolia), Texas blueweed (Helianthus ciliaris), and Devil's claws(Proboscidea louisianica). In other embodiments, the weed comprises aherbicide-resistant ryegrass, for example, a glyphosate resistantryegrass, a paraquat resistant ryegrass, a ACCase-inhibitor resistantryegrass, and a non-selective herbicide resistant ryegrass.

In some embodiments, a plant having the GLYAT sequence disclosed hereinor active variants and fragments thereof is not significantly damaged bytreatment with the glyphosate applied to that plant, whereas anappropriate control plant is significantly damaged by the sametreatment.

Generally, the glyphosate is applied to a particular field (and anyplants growing in it) no more than 1, 2, 3, 4, 5, 6, 7, or 8 times ayear, or no more than 1, 2, 3, 4, or 5 times per growing season.

Thus, methods of the disclosure encompass applications of herbicidewhich are “preemergent,” “postemergent,” “preplant incorporation” and/orwhich involve seed treatment prior to planting.

In one embodiment, methods are provided for coating seeds. The methodscomprise coating a seed with an effective amount of a herbicide or acombination of herbicides (as disclosed elsewhere herein). The seeds canthen be planted in an area of cultivation. Further provided are seedshaving a coating comprising an effective amount of a herbicide or acombination of herbicides (as disclosed elsewhere herein). In otherembodiments, the seeds can be coated with at least one fungicide and/orat least one insecticide and/or at least one herbicide or anycombination thereof.

“Preemergent” refers to a herbicide which is applied to an area ofinterest (e.g., a field or area of cultivation) before a plant emergesvisibly from the soil. “Postemergent” refers to a herbicide which isapplied to an area after a plant emerges visibly from the soil. In someinstances, the terms “preemergent” and “postemergent” are used withreference to a weed in an area of interest, and in some instances theseterms are used with reference to a crop plant in an area of interest.When used with reference to a weed, these terms may apply to only aparticular type of weed or species of weed that is present or believedto be present in the area of interest. While any herbicide may beapplied in a preemergent and/or postemergent treatment, some herbicidesare known to be more effective in controlling a weed or weeds whenapplied either preemergence or postemergence. For example, rimsulfuronhas both preemergence and postemergence activity, while other herbicideshave predominately preemergence (metolachlor) or postemergence(glyphosate) activity. These properties of particular herbicides areknown in the art and are readily determined by one of skill in the art.Further, one of skill in the art would readily be able to selectappropriate herbicides and application times for use with the transgenicplants disclosed herein and/or on areas in which the transgenic plantsare to be planted. “Preplant incorporation” involves the incorporationof compounds into the soil prior to planting.

Thus, improved methods of growing a crop and/or controlling weeds suchas, for example, “pre-planting burn down,” are provided wherein an areais treated with herbicides prior to planting the crop of interest inorder to better control weeds. The disclosure also provides methods ofgrowing a crop and/or controlling weeds which are “no-till” or“low-till” (also referred to as “reduced tillage”). In such methods, thesoil is not cultivated or is cultivated less frequently during thegrowing cycle in comparison to traditional methods; these methods cansave costs that would otherwise be incurred due to additionalcultivation, including labor and fuel costs.

The term “safener” refers to a substance that when added to a herbicideformulation eliminates or reduces the phytotoxic effects of theherbicide to certain crops. One of ordinary skill in the art wouldappreciate that the choice of safener depends, in part, on the cropplant of interest and the particular herbicide or combination ofherbicides. Exemplary safeners suitable for use with the presentlydisclosed herbicide compositions include, but are not limited to, thosedisclosed in U.S. Pat. Nos. 4,808,208; 5,502,025; 6,124,240 and U.S.Patent Application Publication Nos. 2006/0148647; 2006/0030485;2005/0233904; 2005/0049145; 2004/0224849; 2004/0224848; 2004/0224844;2004/0157737; 2004/0018940; 2003/0171220; 2003/0130120; 2003/0078167,the disclosures of which are incorporated herein by reference in theirentirety. The methods of the disclosure can involve the use ofherbicides in combination with herbicide safeners such as benoxacor, BCS(1-bromo-4-[(chloromethyl) sulfonyl]benzene), cloquintocet-mexyl,cyometrinil, dichlormid, 2-(dichloromethyl)-2-methyl-1,3-dioxolane (MG191), fenchlorazole-ethyl, fenclorim, flurazole, fluxofenim, furilazole,isoxadifen-ethyl, mefenpyr-diethyl, methoxyphenone((4-methoxy-3-methylphenyl)(3-methylphenyl)-methanone), naphthalicanhydride (1,8-naphthalic anhydride) and oxabetrinil to increase cropsafety. Antidotally effective amounts of the herbicide safeners can beapplied at the same time as the compounds disclosed herein, or appliedas seed treatments. Therefore an aspect of the present disclosurerelates to the use of a mixture comprising glyphosate, at least oneother herbicide, and an antidotally effective amount of a herbicidesafener.

Seed treatment is useful for selective weed control, because itphysically restricts antidoting to the crop plants. Therefore in oneembodiment, a method for selectively controlling the growth of weeds ina field comprising treating the seed from which the crop is grown withan antidotally effective amount of safener and treating the field withan effective amount of herbicide to control weeds.

An antidotally effective amount of a safener is present where a desiredplant is treated with the safener so that the effect of a herbicide onthe plant is decreased in comparison to the effect of the herbicide on aplant that was not treated with the safener; generally, an antidotallyeffective amount of safener prevents damage or severe damage to theplant treated with the safener. One of skill in the art is capable ofdetermining whether the use of a safener is appropriate and determiningthe dose at which a safener should be administered to a crop.

As used herein, an “adjuvant” is any material added to a spray solutionor formulation to modify the action of an agricultural chemical or thephysical properties of the spray solution. See, for example, Green andFoy (2003) “Adjuvants: Tools for Enhancing Herbicide Performance,” inWeed Biology and Management, ed. Inderjit (Kluwer Academic Publishers,The Netherlands). Adjuvants can be categorized or subclassified asactivators, acidifiers, buffers, additives, adherents, antiflocculants,antifoamers, defoamers, antifreezes, attractants, basic blends,chelating agents, cleaners, colorants or dyes, compatibility agents,cosolvents, couplers, crop oil concentrates, deposition agents,detergents, dispersants, drift control agents, emulsifiers, evaporationreducers, extenders, fertilizers, foam markers, formulants, inerts,humectants, methylated seed oils, high load COCs, polymers, modifiedvegetable oils, penetrators, repellants, petroleum oil concentrates,preservatives, rainfast agents, retention aids, solubilizers,surfactants, spreaders, stickers, spreader stickers, synergists,thickeners, translocation aids, uv protectants, vegetable oils, waterconditioners, and wetting agents.

In addition, methods of the disclosure can comprise the use of aherbicide or a mixture of herbicides, as well as, one or more otherinsecticides, fungicides, nematocides, bactericides, acaricides, growthregulators, chemosterilants, semiochemicals, repellents, attractants,pheromones, feeding stimulants or other biologically active compounds orentomopathogenic bacteria, virus, or fungi to form a multi-componentmixture giving an even broader spectrum of agricultural protection.Examples of such agricultural protectants which can be used in methodsof the disclosure include: insecticides such as abamectin, acephate,acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin,azinphos-methyl, bifenthrin, bifenazate, buprofezin, carbofuran, cartap,chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl,chromafenozide, clothianidin, cyflumetofen, cyfluthrin, beta-cyfluthrin,cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin,diafenthiuron, diazinon, dieldrin, diflubenzuron, dimefluthrin,dimethoate, dinotefuran, diofenolan, emamectin, endosulfan,esfenvalerate, ethiprole, fenothiocarb, fenoxycarb, fenpropathrin,fenvalerate, fipronil, flonicamid, flubendiamide, flucythrinate,tau-fluvalinate, flufenerim (UR-50701), flufenoxuron, fonophos,halofenozide, hexaflumuron, hydramethylnon, imidacloprid, indoxacarb,isofenphos, lufenuron, malathion, metaflumizone, metaldehyde,methamidophos, methidathion, methomyl, methoprene, methoxychlor,metofluthrin, monocrotophos, methoxyfenozide, nitenpyram, nithiazine,novaluron, noviflumuron (XDE-007), oxamyl, parathion, parathion-methyl,permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb,profenofos, profluthrin, pymetrozine, pyrafluprole, pyrethrin,pyridalyl, pyriprole, pyriproxyfen, rotenone, ryanodine, spinosad,spirodiclofen, spiromesifen (BSN 2060), spirotetramat, sulprofos,tebufenozide, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos,thiacloprid, thiamethoxam, thiodicarb, thiosultap-sodium, tralomethrin,triazamate, trichlorfon and triflumuron; fungicides such as acibenzolar,aldimorph, amisulbrom, azaconazole, azoxystrobin, benalaxyl, benomyl,benthiavalicarb, benthiavalicarb-isopropyl, binomial, biphenyl,bitertanol, blasticidin-S, Bordeaux mixture (Tribasic copper sulfate),boscalid/nicobifen, bromuconazole, bupirimate, buthiobate, carboxin,carpropamid, captafol, captan, carbendazim, chloroneb, chlorothalonil,chlozolinate, clotrimazole, copper oxychloride, copper salts such ascopper sulfate and copper hydroxide, cyazofamid, cyflunamid, cymoxanil,cyproconazole, cyprodinil, dichlofluanid, diclocymet, diclomezine,dicloran, diethofencarb, difenoconazole, dimethomorph, dimoxystrobin,diniconazole, diniconazole-M, dinocap, discostrobin, dithianon,dodemorph, dodine, econazole, etaconazole, edifenphos, epoxiconazole,ethaboxam, ethirimol, ethridiazole, famoxadone, fenamidone, fenarimol,fenbuconazole, fencaramid, fenfuram, fenhexamide, fenoxanil,fenpiclonil, fenpropidin, fenpropimorph, fentin acetate, fentinhydroxide, ferbam, ferfurazoate, ferimzone, fluazinam, fludioxonil,flumetover, fluopicolide, fluoxastrobin, fluquinconazole,fluquinconazole, flusilazole, flusulfamide, flutolanil, flutriafol,folpet, fosetyl-aluminum, fuberidazole, furalaxyl, furametapyr,hexaconazole, hymexazole, guazatine, imazalil, imibenconazole,iminoctadine, iodicarb, ipconazole, iprobenfos, iprodione, iprovalicarb,isoconazole, isoprothiolane, kasugamycin, kresoxim-methyl, mancozeb,mandipropamid, maneb, mapanipyrin, mefenoxam, mepronil, metalaxyl,metconazole, methasulfocarb, metiram, metominostrobin/fenominostrobin,mepanipyrim, metrafenone, miconazole, myclobutanil, neo-asozin (ferricmethanearsonate), nuarimol, octhilinone, ofurace, orysastrobin,oxadixyl, oxolinic acid, oxpoconazole, oxycarboxin, paclobutrazol,penconazole, pencycuron, penthiopyrad, perfurazoate, phosphonic acid,phthalide, picobenzamid, picoxystrobin, polyoxin, probenazole,prochloraz, procymidone, propamocarb, propamocarb-hydrochloride,propiconazole, propineb, proquinazid, prothioconazole, pyraclostrobin,pryazophos, pyrifenox, pyrimethanil, pyrifenox, pyrolnitrine,pyroquilon, quinconazole, quinoxyfen, quintozene, silthiofam,simeconazole, spiroxamine, streptomycin, sulfur, tebuconazole,techrazene, tecloftalam, tecnazene, tetraconazole, thiabendazole,thifluzamide, thiophanate, thiophanate-methyl, thiram, tiadinil,tolclofos-methyl, tolyfluanid, triadimefon, triadimenol, triarimol,triazoxide, tridemorph, trimoprhamide tricyclazole, trifloxystrobin,triforine, triticonazole, uniconazole, validamycin, vinclozolin, zineb,ziram, and zoxamide; nematocides such as aldicarb, oxamyl andfenamiphos; bactericides such as streptomycin; acaricides such asamitraz, chinomethionat, chlorobenzilate, cyhexatin, dicofol,dienochlor, etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin,fenpyroximate, hexythiazox, propargite, pyridaben and tebufenpyrad; andbiological agents including entomopathogenic bacteria, such as Bacillusthuringiensis subsp. Aizawai, Bacillus thuringiensis subsp. Kurstaki,and the encapsulated delta-endotoxins of Bacillus thuringiensis (e.g.,Cellcap, MPV, MPVII); entomopathogenic fungi, such as green muscardinefungus; and entomopathogenic virus including baculovirus,nucleopolyhedro virus (NPV) such as HzNPV, AfNPV; and granulosis virus(GV) such as CpGV.

The methods of controlling weeds can further include the application ofa biologically effective amount of a herbicide of interest or a mixtureof herbicides, and an effective amount of at least one additionalbiologically active compound or agent and can further comprise at leastone of a surfactant, a solid diluent or a liquid diluent. Examples ofsuch biologically active compounds or agents are: insecticides such asabamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin,azadirachtin, azinphos-methyl, bifenthrin, binfenazate, buprofezin,carbofuran, chlorfenapyr, chlorfluazuron, chlorpyrifos,chlorpyrifos-methyl, chromafenozide, clothianidin, cyfluthrin,beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin,cyromazine, deltamethrin, diafenthiuron, diazinon, diflubenzuron,dimethoate, diofenolan, emamectin, endosulfan, esfenvalerate, ethiprole,fenothicarb, fenoxycarb, fenpropathrin, fenvalerate, fipronil,flonicamid, flucythrinate, tau-fluvalinate, flufenerim (UR-50701),flufenoxuron, fonophos, halofenozide, hexaflumuron, imidacloprid,indoxacarb, isofenphos, lufenuron, malathion, metaldehyde,methamidophos, methidathion, methomyl, methoprene, methoxychlor,monocrotophos, methoxyfenozide, nithiazin, novaluron, noviflumuron(XDE-007), oxamyl, parathion, parathion-methyl, permethrin, phorate,phosalone, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine,pyridalyl, pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060),sulprofos, tebufenozide, teflubenzuron, tefluthrin, terbufos,tetrachlorvinphos, thiacloprid, thiamethoxam, thiodicarb,thiosultap-sodium, tralomethrin, trichlorfon and triflumuron; fungicidessuch as acibenzolar, azoxystrobin, benomyl, blasticidin-S, Bordeauxmixture (tribasic copper sulfate), bromuconazole, carpropamid, captafol,captan, carbendazim, chloroneb, chlorothalonil, copper oxychloride,copper salts, cyflufenamid, cymoxanil, cyproconazole, cyprodinil,(S)-3,5-dichloro-N-(3-chloro-1-ethyl-1-methyl-2-oxopropyl)-4-methylbenzamide(RH 7281), diclocymet (S-2900), diclomezine, dicloran, difenoconazole,(S)-3,5-dihydro-5-methyl-2-(methylthio)-5-phenyl-3-(phenyl-amino)-4H-imidazol-4-one(RP 407213), dimethomorph, dimoxystrobin, diniconazole, diniconazole-M,dodine, edifenphos, epoxiconazole, famoxadone, fenamidone, fenarimol,fenbuconazole, fencaramid (SZX0722), fenpiclonil, fenpropidin,fenpropimorph, fentin acetate, fentin hydroxide, fluazinam, fludioxonil,flumetover (RPA 403397), flumorf/flumorlin (SYP-L190), fluoxastrobin(HEC 5725), fluquinconazole, flusilazole, flutolanil, flutriafol,folpet, fosetyl-aluminum, furalaxyl, furametapyr (S-82658),hexaconazole, ipconazole, iprobenfos, iprodione, isoprothiolane,kasugamycin, kresoxim-methyl, mancozeb, maneb, mefenoxam, mepronil,metalaxyl, metconazole, metomino-strobin/fenominostrobin (SSF-126),metrafenone (AC375839), myclobutanil, neo-asozin (ferricmethanearsonate), nicobifen (BAS 510), orysastrobin, oxadixyl,penconazole, pencycuron, probenazole, prochloraz, propamocarb,propiconazole, proquinazid (DPX-KQ926), prothioconazole (JAU 6476),pyrifenox, pyraclostrobin, pyrimethanil, pyroquilon, quinoxyfen,spiroxamine, sulfur, tebuconazole, tetraconazole, thiabendazole,thifluzamide, thiophanate-methyl, thiram, tiadinil, triadimefon,triadimenol, tricyclazole, trifloxystrobin, triticonazole, validamycinand vinclozolin; nematocides such as aldicarb, oxamyl and fenamiphos;bactericides such as streptomycin; acaricides such as amitraz,chinomethionat, chlorobenzilate, cyhexatin, dicofol, dienochlor,etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin, fenpyroximate,hexythiazox, propargite, pyridaben and tebufenpyrad; and biologicalagents including entomopathogenic bacteria, such as Bacillusthuringiensis subsp. Aizawai, Bacillus thuringiensis subsp. Kurstaki,and the encapsulated delta-endotoxins of Bacillus thuringiensis (e.g.,Cellcap, MPV, MPVII); entomopathogenic fungi, such as green muscardinefungus; and entomopathogenic virus including baculovirus,nucleopolyhedro virus (NPV) such as HzNPV, AfNPV; and granulosis virus(GV) such as CpGV. Methods of the disclosure may also comprise the useof plants genetically transformed to express proteins (such as Bacillusthuringiensis delta-endotoxins) toxic to invertebrate pests. In suchembodiments, the effect of exogenously applied invertebrate pest controlcompounds may be synergistic with the expressed toxin proteins. Generalreferences for these agricultural protectants include The PesticideManual, 13th Edition, C. D. S. Tomlin, Ed., British Crop ProtectionCouncil, Farnham, Surrey, U.K., 2003 and The BioPesticide Manual, 2^(nd)Edition, L. G. Copping, Ed., British Crop Protection Council, Farnham,Surrey, U.K., 2001.

In certain instances, combinations with other invertebrate pest controlcompounds or agents having a similar spectrum of control but a differentmode of action will be particularly advantageous for resistancemanagement. Thus, compositions of the present disclosure can furthercomprise a biologically effective amount of at least one additionalinvertebrate pest control compound or agent having a similar spectrum ofcontrol but a different mode of action. Contacting a plant geneticallymodified to express a plant protection compound (e.g., protein) or thelocus of the plant with a biologically effective amount of a compound ofthis disclosure can also provide a broader spectrum of plant protectionand be advantageous for resistance management.

Thus, methods of controlling weeds can employ a herbicide or herbicidecombination and may further comprise the use of insecticides and/orfungicides, and/or other agricultural chemicals such as fertilizers. Theuse of such combined treatments disclosed herein can broaden thespectrum of activity against additional weed species and suppress theproliferation of any resistant biotypes.

Methods can further comprise the use of plant growth regulators such asaviglycine, N-(phenylmethyl)-1H-purin-6-amine, ethephon, epocholeone,gibberellic acid, gibberellin A₄ and A₇, harpin protein, mepiquatchloride, prohexadione calcium, prohydrojasmon, sodium nitrophenolateand trinexapac-methyl, and plant growth modifying organisms such asBacillus cereus strain BP01.

C. Methods of Detection

Methods for detecting a GLYAT polypeptide or an active variant orfragment thereof are provided. Such methods comprise analyzing hostcells, plant tissues or plant cells to detect such polypeptides or thepolynucleotides encoding the same. The detection methods can directlyassay for the presence of the GLYAT polypeptide or polynucleotide or thedetection methods can indirectly assay for the sequences by assaying thephenotype of the host cell, plant, plant cell or plant explantexpressing the sequence.

In one embodiment, the GLYAT polypeptide is detected in the plant tissueusing an immunoassay comprising an antibody or antibodies thatspecifically recognizes the GLYAT polypeptide or active variant orfragment thereof. In specific embodiments, the antibody or antibodieswhich are used are raised to a GLYAT polypeptide or active variant orfragment thereof as disclosed herein.

By “specifically or selectively binds” is intended that the bindingagent has a binding affinity for a given GLYAT polypeptide or fragmentor variant disclosed herein, which is greater than 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2% or 1% of the binding affinity for a known GLYAT sequence.One of skill will be aware of the proper controls that are needed tocarry out such a determination

By “antibodies that specifically bind” is intended that the antibodieswill not substantially cross react with another polypeptide. By “notsubstantially cross react” is intended that the antibody or fragmentthereof has a binding affinity for the other polypeptide which is lessthan 10%, less than 5%, or less than 1%, of the binding affinity for theGLYAT polypeptide or active fragment or variant thereof.

In still other embodiments, the GLYAT polypeptide or active variant orfragment thereof can be detected in a host cell or plant tissue bydetecting the various GLYAT polypeptides or active variants andfragments thereof using mass spectrometry. By “detecting” is intendeddetermining the presence or amount of an analyte of interest (i.e., theGLYAT polypeptide) in a test sample. The method of detection is notrestricted and may be either qualitative or quantitative. The term “massspectrometry” or “MS” as used herein generally refer to methods offiltering, detecting, and measuring ions based on their mass-to-chargeratio, or “m/z.” In MS techniques, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry,” which ishereby incorporated by reference in its entirety.

In still other embodiments, the GLYAT polypeptide or active variant orfragment thereof can be detected in a host cell or plant tissue bydetecting the presence of a polynucleotide encoding any of the variousGLYAT polypeptides or active variants and fragments thereof. In oneembodiment, the detection method comprises assaying plant tissue usingPCR amplification.

As used herein, “primers” are isolated polynucleotides that are annealedto a complementary target DNA strand by nucleic acid hybridization toform a hybrid between the primer and the target DNA strand, thenextended along the target DNA strand by a polymerase, e.g., a DNApolymerase. Primer pairs of the disclosure refer to their use foramplification of a target polynucleotide, e.g., by the polymerase chainreaction (PCR) or other conventional nucleic-acid amplification methods.“PCR” or “polymerase chain reaction” is a technique used for theamplification of specific DNA segments (see, U.S. Pat. Nos. 4,683,195and 4,800,159; herein incorporated by reference).

Probes and primers are of sufficient nucleotide length to bind to thetarget DNA sequence and specifically detect and/or identify apolynucleotide encoding a GLYAT polypeptide or active variant orfragment thereof as describe elsewhere herein. It is recognized that thehybridization conditions or reaction conditions can be determined by theoperator to achieve this result. This length may be of any length thatis of sufficient length to be useful in a detection method of choice.Such probes and primers can hybridize specifically to a target sequenceunder high stringency hybridization conditions. Probes and primersaccording to embodiments of the present disclosure may have complete DNAsequence identity of contiguous nucleotides with the target sequence,although probes differing from the target DNA sequence and that retainthe ability to specifically detect and/or identify a target DNA sequencemay be designed by conventional methods. Accordingly, probes and primerscan share about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater sequence identity or complementarity to the targetpolynucleotide.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2.sup.nd ed, vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press: San Diego, 1990.

PCR primer pairs can be derived from a known sequence, for example, byusing computer programs intended for that purpose such as the PCR primeranalysis tool in Vector NTI version 10 (Invitrogen); PrimerSelect(DNASTAR Inc., Madison, Wis.); and Primer (Version 0.5.COPYRGT., 1991,Whitehead Institute for Biomedical Research, Cambridge, Mass.).Additionally, the sequence can be visually scanned and primers manuallyidentified using guidelines known to one of skill in the art.

EXPERIMENTAL Example 1 Obtaining GLYAT Amino Acid Sequence Diversity

Family shuffling of a variant with high catalytic activity towardglyphosate (SEQ ID NO:1) and one with low activity toward aspartate (SEQID NO:2) was initially performed to obtain a library of variants wherethose properties were combined in improved proportions in at least a fewof the individual members. Those variants selected as hits became theparental genes for the next library, and so on. Other methods forintroducing amino acid diversity were also employed such as sitespecific mutagenesis. In one method, sequences related to GLYAT thatwere available in public databases were aligned, and statisticalalgorithms were used to prioritize the diversity available at eachposition. The selected diversity was then randomly toggled into the mostadvanced sequence available with the technique of semi-syntheticshuffling (Ness et al, Nature Biotech 20:1251, 2002). SEQ ID NOs 3-26were thus derived.

In another method, saturation mutagenesis was performed at each positionby the technique of ‘NNK coding’. NNK is a reduced genetic code of 32codons where N represents a 25% mix each of ATGC and K represents a 50%mix each of T and G. By this method, each of the 20 amino acids wasencoded and 2 of 3 stop codons eliminated. The individual substitutionswere combined in recombinant libraries using synthetic or semisyntheticmethods. SEQ ID NOs 27-172 were thus derived.

Example 2 Screen for Variants with Increased Specificity Based on EnzymeActivity Assays

This screen was designed to identify variants with greater specificityfor glyphosate relative to other specific substrates and was based onactivity measurements with crude lysates and with purified proteins.Plasmids containing glyat genes were initially transformed into Top 10E. coli cells (Invitrogen) for plasmid propagation. The plasmids werethen isolated and transformed into BL21(DE3) cells (Invitrogen) forscreening and protein production. Transformed cells were plated onto M9agar containing 1 to 10 mM glyphosate, where only those cells expressinga functional GLYAT enzyme were able to grow. Colonies were picked andarrayed in 96-well or 384-well format for further screening.

Activity with glyphosate and other putative substrates was initiallymeasured with crude supernatants of lysates of E. coli cells expressinga GLYAT variant. Cells were grown in 96-well plates in LB mediumcontaining the inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG).Cells were spun down and lysed with BPER (Pierce). Using a 96-wellliquid handling system, 15 ul of the lysate supernatant were dispensedto each of three 96-well assay plates (Greiner BioOne UV-Star).Reactions were initiated by the addition of 185 ul of reaction mixturecontaining 25 mM Hepes, pH 7.0, 100 mM KCl (when the substrate wasglyphosate) or 20 mM KCl (when the substrate was aspartate or othersubstrate), 10% ethylene glycol, 167 uM acetyl CoA and the aminosubstrate. The rate of utilization of acetyl coenzyme A (AcCoA)supported by each substrate was measured by recording the absorbanceduring a two minute reaction with a Spectramax 384 Plus plate reader(Molecular Devices). If the amino acid present at position 30 (29 ifalanine is absent at position 2) was cysteine, absorbance was monitoredat 235 nM, which directly detects cleavage of the sulfoester bond ofacetyl CoA. If the amino acid present at position 30 (29 if alanine isabsent at position 2) was isoleucine, 0.5 mM5,5′-dithiobis-(2-nitrobenzoic acid), which reacts with the coenzyme Athiol generated in the enzyme catalyzed reaction, was included in thereaction mix and absorbance was monitored at 412 nm. Three separatereactions were run with each lysate, in which the amino substrate was 3mM glyphosate, 0.5 mM glyphosate or 20 mM aspartate. Hits were picked onthe basis of highest activity with 3 mM glyphosate as an indication ofk_(cat), highest value for the ratio of activity at 0.5 mM glyphosate tothat at 3 mM glyphosate as an indicator of K_(M glyph), and highestvalues for the ratio of activity with 3 mM glyphosate to that withaspartate as an indicator of specificity.

To broaden the range of substrates against which to select for activity,substrates with differing structural and chemical properties can becombined. In this example, 20 mM threonine, which has a hydroxyethylside chain, was combined with 20 mM aspartate, which has a carboxymethylside chain, in the screen with crude lysates. The results in Table 2show the improvement in specificity (in this case, reduced activity withL-serine, O-phospho-L-serine and L-threonine) obtained when threoninewas included in the crude lysate activity screen.

The data from the crude lysate activity screen were used to selectapproximately 5 to 10% of the variants that were able to grow on minimalmedium containing glyphosate as potentially having the desired kineticproperties. The final stage of screening was to obtain purified proteinsand perform substrate saturation kinetic analysis.

TABLE 2 Selection of greater specificity of GLYATs using multiplesubstrate assay. The data are micromolar product formed in 30 minutes inreaction mixtures containing 10 mM amino substrate, 0.167 mM AcCoA and0.1 uM enzyme. Selected against activity with aspartate. SEQ ID NO: 2425 11 26 Serine 7.02 6.76 9.78 4.82 P-Serine 6.66 9.15 2.81 1.62 Threo9.66 10.75 9.70 4.73 Selected against activity with aspartate andthreonine SEQ ID NO: 65 29 36 37 83 99 Serine 0.44 1.10 0.65 −0.04 1.290.11 P-Serine 0.08 0.97 0.27 0.47 0.65 0.37 Threo 0.35 0.60 0.42 −0.091.12 0.03

Example 3 Production, Purification and Kinetic Analysis of GLYATProteins

E. coli cultures in LB or 2×YT were grown overnight at 37° C. tosaturation. The cells were used as a 2% inoculum into fresh media. WhenOD reached 0.6, IPTG was added to 0.2 mM and growth was continued for atleast 6 hours. Cells were harvested by centrifugation and stored at −80°C. Cells were lysed with BPER (Pierce) containing 1 mM dithiothreitol,0.2 mg/ml lysozyme, 2 mg/ml protease inhibitor cocktail (Sigma bacterialcell cocktail, P8465) and endonuclease. Lysate supernatants were passedthrough a column of NHS-activated Agarose (Thermo, #26197) derivatizedwith coenzyme A. Columns were washed with 25 mM Hepes, pH 7, containingprogressively higher concentrations of KCl, then eluted with 25 mMHepes, 100 mM KCl, 10% ethylene glycol and 1 mM acetyl CoA. Purity asevaluated by polyacrylamide gel electrophoresis was deemed consistentlyhigh enough to be assumed to be 100%. Protein concentration wasdetermined with the Bradford assay using a large-scale lot of producedGLYAT protein as standard.

Kinetic parameters for various substrates were obtained using a range ofseven substrate concentrations plus a blank containing none of thevaried substrate. The non-varied substrate was present at a saturatingconcentration. Six-microliter aliquots of 50-fold concentrated stocksolutions of the varied substrate were placed in a 96-well assay plate(Greiner BioOne UV-Star). Reactions were started with the addition of294 ul of 25 mM Hepes, pH 7.0, 100 mM KCL, 10% ethylene glycol, thenon-varied substrate and 10 to 200 nM enzyme. Absorbance was monitoredfor one minute with a Spectramax 384 Plus plate reader (MolecularDevices). If the amino acid present at position 30 (29 if alanine isabsent at position 2) was cysteine, absorbance was monitored at 235 nM,which directly detects cleavage of the sulfoester bond of acetyl CoA. Ifthe amino acid present at position 30 (29 if alanine is absent atposition 2) was isoleucine, 0.5 mM 5,5′-dithiobis-(2-nitrobenzoic acid),which reacts with the coenzyme A thiol generated in the enzyme catalyzedreaction, was included in the reaction mix and absorbance was monitoredat 412 nm. Initial rates were processed by the Softmax software toobtain kinetic parameters, using the double-reciprocal transformation.

Kinetic parameters of GLYAT variants identified by the proceduresdescribed in Examples 1-2 are shown in Table 3. k_(cat)/K_(M) withaspartate was reduced from 13.7 min⁻¹ mM⁻¹ in SEQ ID NO:1 to as low as0.1 in novel variants, while specificity (k_(cat)/KMasp/k_(cat)/K_(M glyph)) improved from 1.3% to as low as 0.014%.

TABLE 3 Kinetic analysis of high specificity glyphosateacetyltransferase enzymes derived from SEQ ID NO: 1 and 2. Kineticparameters, Kinetic parameters, glyphosate aspartate Asp as SEQ IDk_(cat), K_(M), k_(cat), K_(M), % of NO: min⁻¹ mM k_(cat)/K_(M) min⁻¹ mMk_(cat)/K_(M) glyph 1 1454 1.36 1080 438 32.9 13.74 1.273 2 271 1.38 19653.2 47.6 1.12 0.574 3 696 0.60 1159 163 35.1 4.67 0.403 4 1613 1.96 827136 78.3 1.74 0.210 5 1019 0.23 4588 215 79.1 2.72 0.059 6 673 2.24 30811 66.8 0.16 0.052 7 428 0.80 532 91 112 0.82 0.154 8 687 0.39 1779 8560.0 1.41 0.079 9 694 0.25 2726 155 78.7 2.00 0.073 10 699 0.37 1901 14989.3 1.69 0.091 11 734 0.44 1684 138 95.9 1.45 0.086 12 425 0.28 1560 8253.9 1.51 0.097 13 516 0.51 1092 87 82.1 1.09 0.100 14 945 0.37 2580 18969.2 2.78 0.108 15 435 0.32 1391 103 72.3 1.43 0.103 16 393 0.69 574 5665.2 0.90 0.157 17 530 0.83 639 95 89.7 1.06 0.165 18 1217 0.73 1714 15753.0 2.96 0.173 19 1016 0.80 1272 148 65.1 2.27 0.178 20 946 0.64 1485165 52.9 3.11 0.209 21 2411 2.59 930 151 72.4 2.08 0.223 22 2527 5.24485 123 104 1.20 0.247 23 427 0.70 613 83.0 80.6 1.03 0.168 24 708 1.31540 65.2 62.6 1.04 0.193 25 678 0.83 832 67.1 112.2 0.60 0.072 26 5600.22 2505 90 104 0.96 0.038 27 563 0.46 1225 72.6 125.7 0.58 0.047 28520 0.54 959 34.5 164.2 0.21 0.022 29 1000 0.28 3831 56.8 116.7 0.480.014 30 255 0.23 1089 17.9 84.5 0.21 0.019 31 530 0.59 904 15 72.9 0.210.023 32 782 0.45 1756 46 103.3 0.44 0.025 33 1222 0.46 2674 78 97.60.80 0.030 34 990 0.68 1458 36 105.1 0.34 0.024 35 544 0.60 904 18 84.40.21 0.023 36 719 0.49 1480 38 146.4 0.26 0.018 37 619 0.68 908 17 88.80.19 0.021 38 388 0.58 665 10 109.2 0.09 0.014 39 637 0.49 1289 31 105.00.30 0.023 40 443 0.17 2686 55 101.7 0.54 0.020 41 356 0.438 812 18.792.9 0.202 0.025 42 290 0.374 776 16.6 81.4 0.204 0.026 43 702 0.883 79526.8 100.0 0.268 0.034 44 671 0.741 906 40.2 113.7 0.353 0.039 45 5460.588 928 26.9 132.1 0.204 0.022 46 654 0.952 687 25.3 155.1 0.163 0.02447 643 0.727 885 22.4 126.7 0.177 0.020 48 962 1.600 601 24.1 141.50.170 0.028 49 566 0.890 636 22.6 97.6 0.232 0.036 50 461 0.405 114239.5 100.5 0.424 0.038 51 360 0.580 620 17.3 95.2 0.182 0.029 52 7090.664 1067 40 129.9 0.309 0.029 53 867 0.912 951 33 135.0 0.242 0.025 54481 1.292 373 10 98.5 0.104 0.028 55 895 0.890 1006 34 102.6 0.330 0.03356 763 0.629 1213 46 129.5 0.355 0.029 57 1131 0.932 1214 47.2 152.10.311 0.026 58 654 0.569 1138 55.1 143.8 0.388 0.034 59 737 0.833 88525.7 96.0 0.268 0.030 60 2735 0.593 4613 168.5 100.3 1.680 0.036 61 9621.147 838 30.7 127.3 0.241 0.029 62 934 0.930 1004 28.5 107.3 0.2660.026 63 1418 1.024 1385 51.8 152.4 0.340 0.025 64 793 0.747 1062 46.2113.0 0.409 0.039 65 1093 0.971 1125 161.7 1237 0.131 0.012 66 718 0.4431620 51 108.4 0.47 0.029 67 702 0.750 936 36 104.4 0.34 0.037 68 10951.026 1067 49 230.6 0.21 0.020 69 986 0.611 1614 43 115.6 0.37 0.023 70985 0.974 1012 33 90.3 0.37 0.036 71 685 1.217 563 15 72.2 0.21 0.038 72465 0.751 619 20 128.3 0.16 0.025 73 519 0.790 657 24 102.5 0.24 0.03674 715 0.797 897 40 157.5 0.25 0.028 75 464 0.636 730 17 99.1 0.17 0.02376 487 0.778 626 22 135.1 0.16 0.026 77 513 0.703 729 20 127 0.16 0.02278 560 0.598 937 32 105 0.31 0.033 79 424 0.473 896 32 112 0.28 0.032 80481 0.783 614 13 91 0.14 0.023 81 459 0.550 834 25 110 0.23 0.027 82 3590.465 772 19 100 0.19 0.025 83 511 0.417 1227 40 111 0.36 0.030 84 3000.28 1079 18.1 67.2 0.27 0.025 85 446 0.564 792 18 97 0.18 0.023 86 4720.519 909 12 86 0.14 0.015 87 555 0.604 919 44 112 0.39 0.042 88 3510.434 808 28 82 0.34 0.042 89 531 1.031 515 15 83 0.18 0.035 90 4900.872 562 12 82 0.15 0.027 91 436 0.831 525 13 82 0.16 0.031 92 4060.479 847 32 108 0.30 0.035 93 500 0.661 757 31 105 0.29 0.039 94 4190.583 719 22 76 0.28 0.040 95 519 0.609 853 33 112 0.29 0.034 96 3840.642 598 24 101 0.24 0.040 97 531 0.461 1151 36 94 0.38 0.033 98 5230.845 618 19 96 0.19 0.031 99 684 0.692 988 34 107 0.32 0.032 100 3130.465 673 12 58 0.21 0.031 101 447 0.621 719 14 112 0.13 0.018 102 4810.205 2346 78.5 94.9 0.83 0.035 103 543 0.191 2844 95.3 102.5 0.93 0.033104 505 0.211 2391 89.9 103.0 0.87 0.036 105 797 0.438 1820 65.6 98.70.66 0.037 106 534 0.323 1654 82.5 108.1 0.76 0.046 107 536 0.313 171477.9 114.8 0.68 0.040 108 513 0.305 1682 87.3 106.8 0.82 0.049 109 4750.28 1696 82.8 117.4 0.71 0.042 110 513 0.329 1560 82.0 99.5 0.82 0.053111 537 0.221 2428 76.0 95.8 0.79 0.033 112 517 0.23 2249 78.0 97.9 0.800.035 113 594 0.245 2422 94.1 109.8 0.86 0.035 114 599 0.225 2668 90.5103.3 0.88 0.033 115 529 0.220 2402 73.9 86.0 0.86 0.036 116 559 0.779718 72.1 93.7 0.77 0.107 117 630 0.536 1176 73.2 94.7 0.77 0.066 118 7100.299 2373 110.9 62.3 1.78 0.075 119 574 0.393 1461 86.2 111.8 0.770.053 120 382 0.331 1154 60.4 115.6 0.52 0.045 121 544 0.161 3431 95 1220.78 0.024 122 523 0.311 1681 91.2 121.7 0.75 0.045 123 454 0.226 200964.4 109.6 0.59 0.029 124 461 0.409 1128 59.4 120.4 0.49 0.044 125 4540.216 2102 81.6 128.0 0.64 0.030 126 475 0.206 2306 75.8 96.6 0.78 0.034127 562 0.202 2784 94.1 96.2 0.98 0.035 128 536 0.198 2706 89.4 88.91.01 0.037 129 346 0.313 1106 60.3 103.7 0.58 0.053 130 642 0.284 2261138.1 172.0 0.80 0.036 131 754 0.434 1737 77.9 137.0 0.57 0.033 132 4740.314 1510 46.1 92.9 0.50 0.033 133 620 0.354 1751 64.8 103.0 0.63 0.036134 911 0.376 2423 74.5 100.2 0.74 0.031 135 703 0.382 1841 49.5 90.10.55 0.030 136 1034 0.503 2055 54.6 78.0 0.70 0.034 137 547 0.297 184160.3 96.6 0.62 0.034 138 312 0.286 1090 45.4 101.2 0.45 0.041 139 6240.681 916 38.9 104.4 0.37 0.041 140 508 0.204 2491 99.9 105.5 0.95 0.038141 473 0.348 1360 60.7 114.1 0.53 0.039 142 402 0.347 1158 61.8 122.40.50 0.044 143 501 0.309 1621 83.2 119.9 0.69 0.043 144 485 0.223 233580.4 125.4 0.64 0.030 145 517 0.303 1706 75.4 104.8 0.72 0.042 146 5700.396 1440 60.3 80.3 0.75 0.052 147 563 0.46 1225 72.6 125.7 0.58 0.047148 609 0.436 1396 68.7 91.6 0.75 0.054 149 855 0.293 2919 121.6 83.71.45 0.050 150 562 0.331 1696 69.6 93.7 0.74 0.044 151 612 0.301 202680.6 95.5 0.85 0.042 152 600 0.406 1477 65 116 0.56 0.038 153 750 0.4611628 75.7 108.0 0.70 0.043 154 485 0.350 1377 61.4 136 0.45 0.033 155709 0.294 2410 121.6 94.8 1.28 0.053 156 867 0.327 2650 155.2 105.7 1.470.055 157 602 0.329 1829 92.1 89.1 1.03 0.056 158 556 0.376 1480 78.588.0 0.89 0.060 159 521 0.344 1513 81.8 106.3 0.77 0.051 160 497 0.3421454 72.2 89.5 0.81 0.056 161 676 0.362 1869 104.0 99.7 1.04 0.056 162532 0.528 1008 72.2 77.3 0.93 0.093 163 775 0.415 1867 92.6 80.3 1.150.062 164 678 0.731 928 123.0 93.2 1.32 0.142 165 4726 2.228 2121 234.883.0 2.83 0.133 166 668 0.478 1404 232 159 1.46 0.104 167 511 0.462 110793.6 112.3 0.83 0.075 168 724 0.497 1456 126.5 118.1 1.07 0.074 169 6850.693 988 174.3 93.0 1.87 0.190 170 410 0.376 1091 51.4 86.0 0.60 0.055171 644 0.409 1573 73.1 108.5 0.67 0.043 172 404 0.376 1074 42.6 89.70.47 0.044

Example 4 Novel Amino Acid Diversity

Amino acid substitutions unique to the high specificity GLYAT proteinsare shown in Table 4. The sequences with the new substitutions werederived through a combination of saturation mutagenesis of an alreadyfit protein (SEQ ID NO:26) and an effective screening cascade as a meansof identifying the improved genes (Examples 1-2). Regions in which novelchanges are concentrated include helix H2a (positions 23 and 25-29) andthe hairpin formed by strands B6 and B7 (positions 124-144). These twostructures are components of the amino substrate binding site. Helix H3,the backbone to which the central beta sheet is anchored, is a regionwhere 24% of the novel substitutions (17 out of 71) disclosed in Table 4are concentrated into 11% of the positions (16 out of 146). None ofthose are near enough to the active site to influence substratespecificity directly. In three cases, multiple novel substitutions werediscovered immediately or nearly adjacent to conserved amino acids shownpreviously to contribute to the catalytic mechanism (Siehl D L et. al.,J Biol Chem, 282:11446, 2007). The catalytic amino acids for which thisis the case are R21, R111, H138 and the nearby positions with multiplesubstitutions are 23, 112 and 137. The diverse amino acids at thesepositions may influence the orientation of the neighboring catalyticamino acids so as to selectively optimize catalysis with the preferredsubstrate, glyphosate.

Helix H1 (positions 9-19), strand B3 (positions 50-58) and helix H4(positions 113-120) are regions where screening for substratespecificity resulted in the elimination of most of the diversitydiscovered earlier, with most amino acids reverting to those present innative GLYAT. See FIG. 2 for structural details.

In comparing the amino acid sequences of native GLYAT (SEQ ID NO:173),SEQ ID NO:1 and one of the highest specificity variants (SEQ ID NO:29),a motif is revealed as shown in Table 5; this motif is represented bySEQ ID NO:174. In reducing activity with aspartate from 13.7 min⁻¹ mM⁻¹with SEQ ID NO:1 to 0.48 with SEQ ID NO:29, five amino acids in helixH2a-H2b are changed to ones with larger, mostly aliphatic side chains.Most significantly, C291 and F31Y fill a spatial vacancy that molecularmodels suggest could be occupied by the amino group of L-amino acids andother branched compounds, thus improving specificity for the linearglyphosate molecule. The other up-sizing mutations may function torestrict access of water molecules into the active site, which mayincrease specificity by disallowing formation of water wires betweenunwanted substrates and the catalytic base, H138. Additionally,according to the Columbus equation (the attractive force of oppositecharges is inversely related to the dielectric constant of the medium),increased hydrophobicity in the active site would strengthen the chargeinteractions between glyphosate and the side chains that ligate it (R21,R73, R111 and H138). Thus, in some embodiments, the motif VIMYETDLL (SEQID NO:174) is a determinant for highest-specificity GLYAT proteins. SEQID NOs:26-115 and 119-172 contain the motif (SEQ ID NO:174).

TABLE 4New sequence diversity generated in high specificity GLYAT variants.Numbering is based on SEQ ID NO: 2. New New New Position SubstitutionPosition Substitution Position Substitution  1  51 101 ACLI  2  52 102 3 SC  53 103  4  54 104  5  55 105 T  6 M  56 106  7  57 107  8 G  58 G108  9  59 C 109 FQ 10  60 L 110 11  61 111 12  62 112 VLM 13  63 SR 11314  64 114 15  65 115 16  66 116 17  67 C 117 18  68 V 118 19  69 119 20 70 120 21  71 121 22  72 122 23 GRK  73 123 24  74 124 25 S  75 A 125 S26 F  76 126 27 SR  77 127 28 RK  78 128 GTARC 29 I  79 G 129 30  80 130W 31 W  81 131 QR 32  82 132 Y 33  83 N 133 K 34  84 134 35  85 R 135 36 86 136 37  87 A 137 EARS 38  88 R 138 39 C  89 RN 139 40  90 V 140 41 S 91 M 141 42 H  92 142 VC 43  93 143 44  94 144 45  95 Q 145 46 C  96 D146 47  97 48 A  98 M 49  99 VAK 50 100 A

TABLE 5 Generation of a motif for high activity and specificity inGLYAT enzymes. kcat/Km asp, % Variant 28 29 30 31 32 33 34 35 36 glyphasp glyph SEQ ID NO: 173 A C M Y E T D L L 0.27   0.15 55.6   SEQ ID NO: 29 V I M Y E T D L L 3830   0.48 0.013 SEQ ID NO: 1 A C M F ES D L T 1080 13.7 1.27 

Example 5 Efficacy of High Specificity Glyat Genes in Multiple Crops

Glyphosate acetyltransferase (glyat) genes selected for high specificityto glyphosate were introduced into multiple plant species and evaluatedfor both efficacy and phenotype in the greenhouse and field.Agrobacterium-mediated transformation of maize was performed usingwell-known transformation procedures. All T0 plants are transplanted tosoil and grown in a greenhouse under standard conditions. T0 plants aretypically sprayed about 2 weeks after transplanting with 1680 g ae/ha(2×) commercial glyphosate formulation such as Touchdown™ or RoundUpWeatherMax™. T1 plants are often evaluated after a 3360 g ae/ha (4×)glyphosate application. Rates of glyphosate for canola were 1350 g ae/ha(2×) for both T0 and T1. Field rates ranged from 0× to 8× glyphosatetreatment at stages from V3 to reproductive stage. All applicationstypically included ammonium sulfate. As listed in Table 6, transformedevents in all crops showed excellent efficacy with the glyat genes whenexpressed with Ubiquitin promoters for moderate constitutive expressionin all tissues. No deviations in phenotype from untransformed plantswere observed in advanced events.

TABLE 6 High specificity glyphosate acetyltransferase genes conferrobust glyphosate tolerance in multiple crop species. % T0 events % T1events Tested Field with low/no with low/no efficacy herbicide herbicidewith <10% response response herbicide glyat Gene Crop (2X rate) (4Xrate) response SEQ ID NO: 3 Maize 86% ND 4X glyphosate at V4 SEQ ID NO:19 Maize 86% 100%  ND SEQ ID NO: 11 Maize 70% 99% 4X glyphosate at V4SEQ ID NO: 26 Maize 52% 100%  ND SEQ ID NO: 25 Maize 64% ND ND SEQ IDNO: 132 Maize 54% ND ND SEQ ID NO: 134 Maize 58% ND ND SEQ ID NO: 3 Soy65% 34% 4X glyphosate at V3 SEQ ID NO: 4 Soy 50% 13% 4X glyphosate at V3SEQ ID NO: 19 Soy 75% 54% 4X glyphosate at V3 SEQ ID NO: 11 Soy 69% 46%4X glyphosate at V3 SEQ ID NO: 9 Soy 89% 30% ND SEQ ID NO: 6 Soy 85% 23%ND SEQ ID NO: 26 Soy 100%  ND ND SEQ ID NO: 25 Soy 82% ND ND SEQ ID NO:132 Soy 100%  ND ND SEQ ID NO: 3 Canola 93% 98% 8X at 3-5 leaf stage SEQID NO: 19 Canola 95% 97% ND SEQ ID NO: 11 Canola 92% 98% 4X at 3-5 leafstage SEQ ID NO: 3 Rice 100%  100%  8X at V6 SEQ ID NO: 11 Rice 100% 100%  ND ND; No data to date

Example 6 Reduced Accumulation of N-Acetyl Amino Acids in TransgenicPlants Expressing High-Specificity Glyat Genes

Some amino acids, primarily aspartate (Asp) and glutamate (Glu), can actas substrate for glyphosate acetyltransferase enzymes and becomeacetylated, resulting in in planta accumulation of N-acetyl amino acids.While accumulation of N-acetyl amino acids has not been reported to posea safety concern (see e.g., Delaney et al. 2008. Food Chem Toxicol46:2023-2034; Harper et al. 2009. Food Chem Toxicol 47:2723-2729),increased glyphosate acetyltransferase enzyme specificity will improveefficiency. Tissue samples from plants expressing glyat genes wereprepared and analyzed for N-acetyl amino acids by the method describedin Hession A O, Esrey E G, Croes R A, Maxwell C A (2008)N-Acetylglutamate and N-Acetylaspartate in Soybeans (Glycine max L.),Maize (Zea maize L.), and Other Foodstuffs. Journal of Agricultural andFood Chemistry 56:9121-9126. Plants expressing high-specificity GLYATenzymes, e.g. SEQ ID NO: 11 accumulated substantially lower levels ofNA-Asp than plants expressing SEQ ID NO:1 as shown in Table 7. Thisreduction of acetylated amino acids is an indication of the improvedsubstrate specificity of the enzyme, and is consistent with in vitroenzyme analysis.

TABLE 7 Crop plants expressing high-specificity GLYAT enzymes accumulatereduced average levels of NA-Asp in leaf and seed tissue as compared toSEQ ID NO: 1 when both genes are driven by the same promotercombination. NA-Asp Fold glyat Gene Sample type (ug/g) reduction CropSEQ ID NO: 1 Leaf (Tn) 303 Control maize SEQ ID NO: 11 leaf (T0) 1323X   maize SEQ ID NO: 1 Leaf (bulk) T3 23,575 Control canola SEQ ID NO:3 Leaf (bulk) T3 7,061  3.3X canola SEQ ID NO: 11 Leaf (bulk) T1 1,95412.0X canola SEQ ID NO: 1 Seed (bulk) 2,034 Control canola T3 SEQ ID NO:3 Seed (bulk) 767  2.6X canola T3 SEQ ID NO: 1 Leaf (T0) 879 Controlrice SEQ ID NO: 3 Leaf (T0) 45 19.5X rice SEQ ID NO: 11 Leaf (T0) 6147X   rice

Example 7 Glyphosate and Glufosinate Tolerance of Shuffled GLYATVariants in Molecular Stacks with moPAT in Corn

Maize hybrids containing events with different GLYAT shuffled variantsin a molecular stack with moPAT were planted at five midwest USAregulated transgenic research locations. The plots were sprayed withglyphosate at 3.0 lb ae/a (4×) at the V4 and V8 growth stages and withglufosinate at 0.8 lb ae/a (2×) at the V4 growth stage. Plots wereevaluated for crop response at 7 and 14 days after treatment (DAT) on a0-100 scale, where O=no difference between treated and untreated controlplots and 100=complete plant death. Data represent the crop responseaveraged over location, replication, and events within a construct(Table 8).

TABLE 8 Crop response to glyphosate or glufosinate averaged overlocation, replication, and events SEQ Gly 4X V4 Gly 4X V8 Glu 2X V4 IDDev 7 14 7 14 7 14 Construct NO: gene DAT DAT DAT DAT DAT DAT A 6 3 4952 52 56 6 3 B 5 3 12 7 11 12 5 3 C 5 4 12 8 11 12 5 4 D 11 3 11 6 10 104 2 E 11 4 30 21 33 52 10 7 F 3 3 28 21 29 29 4 4 G 3 4 27 18 26 25 6 3H 3 5 27 18 25 25 5 3

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisdisclosure pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the disclosure has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

1-7. (canceled)
 8. A nucleic acid construct comprising an isolated orrecombinant polynucleotide selected from the group consisting of: a) anisolated or recombinant polynucleotide comprising a nucleotide sequenceencoding a polypeptide, wherein said polypeptide hasglyphosate-N-acetyltransferase (GLYAT) activity and comprises an aminoacid sequence comprising SEQ ID NO:174; and b) an isolated orrecombinant polynucleotide comprising a nucleotide sequence encoding apolypeptide, wherein said polypeptide has glyphosate-N-acetyltransferase(GLYAT) activity and comprises an amino acid sequence having at least60%, 70%, 80%, 90% or 95% sequence identity across the full length ofany one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 116, 117, or
 118. 9. The nucleicacid construct of claim 8, further comprising a promoter operably linkedto said polynucleotide.
 10. The nucleic acid construct of claim 9,further comprising the 35S enhancer or an active variant or fragmentthereof operably linked to said promoter.
 11. A cell comprising thenucleic acid construct of claim 8, wherein said polynucleotide isheterologous to the cell.
 12. The cell of claim 11, wherein said cell isa plant cell.
 13. The plant cell of claim 12, wherein saidpolynucleotide or said nucleic acid construct is stably incorporatedinto the genome of said plant cell.
 14. The plant cell of claim 12,wherein said plant cell is from a monocot.
 15. The monocot plant cell ofclaim 14, wherein said monocot is maize, wheat, rice, barley, sugarcane,sorghum, turf grass, or rye.
 16. The plant cell of claim 12, whereinsaid plant cell is from a dicot.
 17. The dicot plant cell of claim 16,wherein the dicot is soybean, Brassica, sunflower, cotton, canola, oralfalfa.
 18. A plant comprising the plant cell of claim
 12. 19. Anexplant comprising the plant cell of claim
 12. 20. The plant of claim18, the explant of claim 19, or the plant cell of claim 12, wherein theplant, explant or plant cell further comprises at least one polypeptideimparting tolerance to an additional herbicide.
 21. The plant, explant,or plant cell of claim 20, wherein said at least one polypeptideimparting tolerance to an additional herbicide comprises: (a) asulfonylurea-tolerant acetolactate synthase; (b) animidazolinone-tolerant acetolactate synthase; (c) a4-hydroxyphenylpyruvate dioxygenase; (d) a phosphinothricin acetyltransferase; (e) a protoporphyrinogen oxidase. (f) an auxin tolerancegene; (g) a P450 polypeptide; (h) an acetyl coenzyme A carboxylase(ACCase) (i) a dicamba monooxygenase; or (j) an aryloxyalkanoatedioxygenase.
 22. The plant, explant, or plant cell of claim 20, whereinsaid at least one polypeptide imparting tolerance to an additionalherbicide comprises a high resistance allele of acetolactate synthase(HRA).
 23. The plant, explant, or plant cell of claim 20, wherein theplant, explant or plant cell comprises at least one additionalpolypeptide imparting tolerance to glyphosate.
 24. The plant, explant orplant cell of claim 23, wherein said at least one polypeptide comprisesa glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase or aglyphosate-tolerant glyphosate oxido-reductase.
 25. A transgenic seedproduced by the plant of claim
 18. 26-32. (canceled)
 33. A method ofproducing a glyphosate tolerant plant cell comprising transforming aplant cell with the nucleic acid construct of claim
 8. 34. The method ofclaim 33, wherein said method further comprises regenerating atransgenic plant from said plant cell.
 35. The method of claim 33,wherein said transforming the plant cell results in the stableintegration of the polynucleotide into the genome of the plant cell. 36.A method for controlling weeds in a field containing a crop comprising:(a) planting an area of cultivation with a crop comprising thetransgenic seed of claim 25 or the plant of claim 18; and, (b) applyingto the area of cultivation, the crop or a weed in the field a sufficientamount of glyphosate or a combination of herbicides including glyphosateto control weeds without significantly affecting the crop. 37-40.(canceled)
 41. The plant of claim 18, the explant of claim 19, or theplant cell of claim 12, wherein the plant, explant or plant cell furthercomprises at least one polypeptide having pesticidal and/or insecticidalactivity.
 42. The nucleic acid construct of claim 8, wherein theisolated or recombinant polynucleotide of claim 1(a) encodes apolypeptide comprising an amino acid sequence having at least 60%, 70%,80%, 90% or 95% sequence identity across the full length of any one ofSEQ ID NOS: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, or 172.